U.S. patent application number 14/386242 was filed with the patent office on 2015-03-19 for methods for treating and monitoring the status of cancer.
This patent application is currently assigned to STEMLINE THERAPEUTICS, INC.. The applicant listed for this patent is STEMLINE THERAPEUTICS, INC.. Invention is credited to Ivan Bergstein, Christopher Brooks, Thomas P. Cirrito.
Application Number | 20150079135 14/386242 |
Document ID | / |
Family ID | 49223299 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079135 |
Kind Code |
A1 |
Cirrito; Thomas P. ; et
al. |
March 19, 2015 |
METHODS FOR TREATING AND MONITORING THE STATUS OF CANCER
Abstract
Provided herein are methods for treating cancer in a subject
comprising administering to the subject a therapeutically effective
amount of a peptide derived from the EphA2 protein and/or the
IL-13R.alpha.2 protein and monitoring the amount of cancer stem
cells in said subject. Also provided herein are methods for
monitoring the efficacy of an EphA2 peptide-based cancer treatment
or an IL-13R.alpha.2 peptide-based cancer treatment in a patient
with cancer, comprising monitoring the amount of cancer stem cells
in said subject prior to, during, and/or following cancer treatment
of a patient.
Inventors: |
Cirrito; Thomas P.; (Long
Island City, NY) ; Bergstein; Ivan; (New York,
NY) ; Brooks; Christopher; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STEMLINE THERAPEUTICS, INC. |
New York |
NY |
US |
|
|
Assignee: |
STEMLINE THERAPEUTICS, INC.
New York
NY
|
Family ID: |
49223299 |
Appl. No.: |
14/386242 |
Filed: |
March 19, 2013 |
PCT Filed: |
March 19, 2013 |
PCT NO: |
PCT/US2013/032936 |
371 Date: |
September 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61612826 |
Mar 19, 2012 |
|
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|
Current U.S.
Class: |
424/400 ;
424/139.1; 424/174.1; 424/277.1; 424/93.7; 424/94.5; 435/29;
435/7.23; 514/19.3 |
Current CPC
Class: |
C12Y 207/10001 20130101;
G01N 2333/7155 20130101; G01N 2333/70596 20130101; A61P 35/00
20180101; C07K 16/40 20130101; A61K 38/45 20130101; G01N 2800/52
20130101; A61K 2039/505 20130101; A61P 37/04 20180101; G01N
33/57407 20130101; A61P 25/00 20180101; C07K 16/2866 20130101; C12N
9/12 20130101; G01N 2333/91205 20130101; A61K 38/1793 20130101 |
Class at
Publication: |
424/400 ;
424/94.5; 424/139.1; 424/174.1; 424/277.1; 514/19.3; 424/93.7;
435/29; 435/7.23 |
International
Class: |
C07K 16/40 20060101
C07K016/40; G01N 33/574 20060101 G01N033/574; A61K 38/17 20060101
A61K038/17; C12N 9/12 20060101 C12N009/12; A61K 38/45 20060101
A61K038/45; C07K 16/28 20060101 C07K016/28 |
Claims
1. A method for treating, preventing, or managing cancer in a
subject in need thereof comprising (i) administering to said
subject a composition comprising an EphA2 peptide or an
IL-13R.alpha.2 peptide and (ii) measuring the amount of cancer stem
cells in said subject.
2. The method of claim 1, wherein the amount of cancer stem cells
in said subject is reduced and or does not increase.
3. A method for monitoring the efficacy of an EphA2 peptide-based
cancer therapy or an IL-13R.alpha.2 peptide-based cancer therapy in
a patient with cancer, the method comprising: (a) measuring the
amount of cancer stem cells in or from the patient before and
following the administration of the cancer therapy; and (b)
comparing the amount of cancer stem cells in or from the patient
before the administration of the cancer therapy to the amount of
cancer stem cells in or from the patient following the
administration of the cancer therapy; wherein the cancer therapy is
determined to be efficacious if the amount cancer stem cells in or
from the patient following the administration of the cancer therapy
is equivalent to or less than the amount of cancer stem cells in or
from the patient before the administration of the cancer
therapy.
4. The method of claim 1, wherein the amount of cancer stem cells
is determined using a biological fluid, a bone marrow biopsy, a
tumor biopsy, a normal tissue biopsy from the patient.
5. The method of claim 1 or 3, wherein the amount of cancer stem
cells is determined by using an immunoassay, a flow cytometer,
immunohistochemistry, a sphere forming assay, an immunocompromised
mouse in vivo engraftment assay, imaging, or by culturing a sample
obtained from the patient and quantitating the cells in an in vitro
assay.
6-14. (canceled)
15. The method of claim 5, wherein said imaging MRI, PET, FDG-PET,
CT scan, or X-RAY.
16. The method of claim 1, further comprising comparing the amount
of cancer stem cells in a sample obtained from the patient to the
amount of cancer stem cells in a reference sample, or to a
predetermined reference range, wherein a stabilization or a
decrease in the amount of cancer stem cells in the sample relative
to the reference sample, or to a predetermined reference range,
indicates that the method is effective.
17. The method of claim 1, wherein said cancer stem cells are
associated with a brain cancer tumor.
18. (canceled)
19. (canceled)
20. The method of claim 1, wherein said EphA2 peptide is a T cell
epitope of EphA2.
21. The method of claim 20, wherein said T cell epitope of EphA2
induces an immune response in a subject.
22. The method of claim 1, wherein said EphA2 peptide comprises or
consists of SEQ ID NO.:1.
23. (canceled)
24. The method of claim 1, wherein said composition is cell
free.
25. (canceled)
26. The method of claim 1, wherein said EphA2 peptide or said
IL-13R.alpha.2 peptide in said composition is loaded on dendritic
cells.
27. The method of claim 1, wherein said composition is administered
to the subject subcutaneously or intra-nodally.
28. A method of treating cancer in a subject in need thereof,
comprising administering to said subject a therapeutically
effective amount of a compound that targets EphA2 or
IL-13R.alpha.2.
29. The method of claim 28, wherein said compound is an antibody
that binds to EphA2 or is an antibody that binds to
IL-13R.alpha.2.
30. (canceled)
31. The method of claim 29, wherein said cancer is brain
cancer.
32. (canceled)
33. The method of claim 28, wherein said method further comprises
measuring the amount of cancer stem cells in said subject.
34-46. (canceled)
47. A method of improving the targeting of cancer stem cells with a
cancer vaccine comprising (i) determining the binding motif of a
Class I or Class II epitope from EphA2, and making substitutions in
the amino acid sequence such that the modified peptides are able to
induce an immune response that is at least as effective at killing
cancer stem cells as the wild type peptide; or (ii) determining the
binding motif of a Class I or Class II epitope from IL-13R.alpha.2,
and making substitutions in the amino acid sequence such that the
modified peptides are able to induce an immune response that is at
least as effective at killing cancer stem cells as the wild type
peptide.
48-51. (canceled)
52. The method of claim 1, wherein the amount of cancer stem cells
is measured by determining the amount of EphA2-expressing cancer
stem cells or IL-13R.alpha.2-expressing cancer stem cells, or
CD133-expressing cancer stem cells.
53-61. (canceled)
Description
1. INTRODUCTION
[0001] Provided herein are methods for treating cancer in a subject
comprising administering to the subject a therapeutically effective
amount of a peptide derived from the EphA2 protein or the
IL-13R.alpha.2 protein and monitoring the amount of cancer stem
cells in said subject. Also provided herein are methods for
monitoring the efficacy of an EphA2 peptide-based cancer treatment
or an IL-13R.alpha.2 peptide-based cancer treatment in a patient
with cancer, comprising monitoring the amount of cancer stem cells
in said subject prior to, during, and/or following cancer treatment
of a patient.
2. BACKGROUND
[0002] Conventional cancer therapies include surgery, chemotherapy,
and radiation therapy. Despite the existence of these therapies, as
well as the significant amount of scientific and medical research
dedicated annually to uncovering cancer therapeutics, cancer
remains one of the leading causes of mortality and morbidity
worldwide today. As such, there remains a need for new and
effective cancer therapeutics, as well as methods for monitoring
the efficacy of existing and new cancer therapeutics.
3. SUMMARY
[0003] In one aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a peptide derived from the
EphA2 protein and monitoring the amount of cancer stem cells in
said subject. In a specific embodiment, the amount of cancer stem
cells in said subject that express EphA2 is measured. In another
specific embodiment, the amount of cancer stem cells in said
subject that express CD133 is measured. In another specific
embodiment, the amount of cancer stein cells in said subject that
express EphA2 and CD 133 is measured.
[0004] In another aspect, provided herein are methods for
monitoring the efficacy of an EphA2 peptide-based cancer treatment
(i.e., a treatment or therapy that comprises administration of a
peptide derived from EphA2) for a patient with cancer, comprising
monitoring the amount of cancer stem cells in said subject prior
to, during, and/or following the cancer treatment of a patient. In
a specific embodiment, the amount of cancer stem cells in said
subject that express EphA2 is measured. In another specific
embodiment, the amount of cancer stem cells in said subject that
express CD133 is measured. In another specific embodiment, the
amount of cancer stem cells in said subject that express EphA2 and
CD133 is measured.
[0005] In another aspect, provided herein are methods for treating
cancer comprising administering a T cell epitope of EphA2 that
targets cancer stem cells, wherein said epitope is sufficient to
induce an immune response in a patient with cancer.
[0006] In another aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a compound that targets the
EphA2 protein, wherein said compound is capable of killing and/or
preventing the differentiation of cancer stem cells that express
the EphA2 protein. In a specific embodiment, the compound is an
antibody that specifically binds EphA2.
[0007] In another aspect, provided herein are methods of improving
the targeting of cancer stem cells with a cancer vaccine comprising
determining the binding motif of a Class I or Class II epitope from
EphA2, and making substitutions in the amino acid sequence such
that the modified peptides are able to induce an immune response
that is at least as effective at killing cancer stem cells as the
wild type peptide.
[0008] In another aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a peptide derived from the
IL-13R.alpha.2 protein and monitoring the amount of cancer stem
cells in said subject. In a specific embodiment, the amount of
cancer stem cells in said subject that express IL-13R.alpha.2 is
measured. In another specific embodiment, the amount of cancer stem
cells in said subject that express CD133 is measured. In another
specific embodiment, the amount of cancer stem cells in said
subject that express IL-13R.alpha.2 and CD133 is measured.
[0009] In another aspect, provided herein are methods for
monitoring the efficacy of an IL-13R.alpha.2 peptide-based cancer
treatment (i.e., a treatment or therapy that comprises
administration of a peptide derived from IL-13R.alpha.2) for a
patient with cancer, comprising monitoring the amount of cancer
stem cells in said subject prior to, during, and/or following the
cancer treatment of a patient. In a specific embodiment, the amount
of cancer stem cells in said subject that express IL-13R.alpha.2 is
measured. In another specific embodiment, the amount of cancer
stein cells in said subject that express CD133 is measured. In
another specific embodiment, the amount of cancer stem cells in
said subject that express IL-13R.alpha.2 and CD133 is measured.
[0010] In another aspect, provided herein are methods for treating
cancer comprising administering a T cell epitope of IL-13R.alpha.2
that targets cancer stem cells, wherein said epitope is sufficient
to induce an immune response in a patient with cancer.
[0011] In another aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a compound that targets the
IL-13R.alpha.2 protein, wherein said compound is capable of killing
and/or preventing the differentiation of cancer stem cells that
express the IL-13R.alpha.2 protein. In a specific embodiment, the
compound is an antibody that specifically binds IL-13R.alpha.2.
[0012] In another aspect, provided herein are methods of improving
the targeting of cancer stem cells with a cancer vaccine comprising
determining the binding motif of a Class I or Class II epitope from
IL-13R.alpha.2, and making substitutions in the amino acid sequence
such that the modified peptides are able to induce an immune
response that is at least as effective at killing cancer stem cells
as the wild type peptide.
3.1 Definitions
[0013] As used herein, the terms "about" or "approximately" when
used in conjunction with a number refer to any number within 1, 5
or 10% of the referenced number.
[0014] As used herein, the term "agent" refers to any molecule,
compound, and/or substance that can be used in or in combination
with a method treatment described herein. The term agent includes,
without limitation, proteins, immunoglobulins (e.g., multi-specific
Igs, single chain Igs, Ig fragments, polyclonal antibodies and
their fragments, monoclonal antibodies and their fragments),
peptides (e.g., peptide receptors, selectins), binding proteins,
biologics, chemospecific agents, chemotoxic agents, anti-angiogenic
agents, and small molecule drugs.
[0015] As used herein, the term "peptide" refers to a polymer of
amino acids linked by amide bonds as is known to those of skill in
the art. A peptide can be a polymer of 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100
or more amino acids linked by covalent amide bonds. In some
embodiments, the peptide is a polymer of 6 to 8, 8 to 10, 10 to 15,
10 to 20, 10 to 25, 10 to 30, 10 to 40, 10 to 50, or 25 to 25 amino
acids linked by covalent amide bonds. In certain embodiments, the
peptide is a polymer of 50 to 65, 50 to 75, 50 to 85, 50 to 95, 50
to 100, 75 to 100 amino acids linked by covalent amide bonds. As
used herein, the term can refer to a single peptide chain linked by
covalent amide bonds. The term can also refer to multiple peptide
chains associated by non-covalent interactions such as ionic
contacts, hydrogen bonds, Van der Waals contacts and hydrophobic
contacts. Those of skill in the art will recognize that the term
includes peptides that have been modified, for example by
post-translational processing such as signal peptide cleavage,
disulfide bond formation, glycosylation (e.g., N-linked
glycosylation), protease cleavage and lipid modification (e.g.
S-palmitoylation).
[0016] As used herein, the terms "purified" and "isolated" when
used in the context of a peptide that is obtained from a natural
source, e.g., cells, refers to a peptide which is substantially
free of contaminating materials from the natural source, e.g., soil
particles, minerals, chemicals from the environment, and/or
cellular materials from the natural source, such as but not limited
to cell debris, cell wall materials, membranes, organelles, the
bulk of the nucleic acids, carbohydrates, proteins, and/or lipids
present in cells. Thus, a peptide that is isolated includes
preparations of a polypeptide having less than about 30%, 20%, 10%,
5%, 2%, or 1% (by dry weight) of cellular materials and/or
contaminating materials. As used herein, the terms "purified" and
"isolated" when used in the context of a peptide that is chemically
synthesized refers to a peptide which is substantially free of
chemical precursors or other chemicals which are involved in the
syntheses of the polypeptide.
[0017] As used herein, the term "nucleic acid" is intended to
include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid can be single-stranded or
double-stranded.
[0018] As used herein, the term "therapeutically effective regimen"
refers to a regimen for dosing, timing, frequency, and duration of
the administration of one or more therapies for the treatment
and/or management of cancer or a symptom thereof.
[0019] As used herein, the terms "subject" or "patient" are used
interchangeably to refer to an animal (e.g., birds, reptiles, and
mammals). In a specific embodiment, a subject is a bird. In another
embodiment, a subject is a mammal including a non-primate (e.g., a
camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat,
and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
In certain embodiments, a subject is a non-human animal. In some
embodiments, a subject is a farm animal or pet. In another
embodiment, a subject is a human. In another embodiment, a subject
is a human infant. In another embodiment, a subject is a human
toddler. In another embodiment, a subject is a human child. In
another embodiment, a subject is a human adult. In another
embodiment, a subject is an elderly human.
[0020] As used herein, the term "brain cancer" refers to a tumor
located inside the cranium or in the central spinal canal. Brain
cancer refers to both primary tumors (i.e., tumors that originate
in the intracranial sphere or the central spinal canal) and
secondary tumors (i.e., tumors that invaded the intracranial sphere
or the central spinal canal after originating from tumors primarily
located in other organs).
[0021] As used herein, the terms "therapies" and "therapy" can
refer to any protocol(s), method(s), composition(s),
formulation(s), and/or agent(s) that can be used in the prevention
or treatment of brain cancer or a disease or symptom associated
therewith. In certain embodiments, the terms "therapies" and
"therapy" refer to biological therapy, supportive therapy, and/or
other therapies useful in treatment or prevention of cancer or a
disease or symptom associated therewith known to one of skill in
the art.
[0022] As used herein, the term "therapeutically effective amount"
refers to the amount of a therapy that is sufficient to result in
the prevention of the development, recurrence, or onset of cancer
and one or more symptoms thereof, to enhance or improve the
prophylactic effect(s) of another therapy, reduce the severity, the
duration of cancer, ameliorate one or more symptoms of cancer,
prevent the advancement of cancer, cause regression of cancer,
and/or enhance or improve the therapeutic effect(s) of another
therapy. In one embodiment, the amount of a therapeutically
effective amount is effective to achieve one, two, three, or more
results following the administration of one, two, three or more
therapies: (1) a stabilization, reduction or elimination of the
cancer stein cell population; (2) a stabilization, reduction or
elimination in the cancer cell population; (3) a stabilization or
reduction in the growth of a tumor or neoplasm; (4) an impairment
in the formation of a tumor; (5) eradication, removal, or control
of primary, regional and/or metastatic cancer; (6) a reduction in
mortality; (7) an increase in disease-free, relapse-free,
progression-free, and/or overall survival, duration, or rate; (8)
an increase in the response rate, the durability of response, or
number of patients who respond or are in remission; (9) a decrease
in hospitalization rate, (10) a decrease in hospitalization
lengths, (11) the size of the tumor is maintained and does not
increase or increases by less than 10%, preferably less than 5%,
preferably less than 4%, preferably less than 2%, (12) an increase
in the number of patients in remission, (13) an increase in the
length or duration of remission, (14) a decrease in the recurrence
rate of cancer, (15) an increase in the time to recurrence of
cancer, and (16) an amelioration of cancer-related symptoms and/or
quality of life.
[0023] As used herein, the term "in combination" in the context of
the administration of a therapy to a subject refers to the use of
more than one therapy (e.g., prophylactic and/or therapeutic). The
use of the term "in combination" does not restrict the order in
which the therapies (e.g., a first and second therapy) are
administered to a subject. A therapy can be administered prior to
(e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1
hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72
hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 8 weeks, or 12 weeks before), concomitantly with, or
subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes,
45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours,
48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a
second therapy to a subject which had, has, or is susceptible to
brain cancer. The therapies are administered to a subject in a
sequence and within a time interval such that the therapies can act
together. In a particular embodiment, the therapies are
administered to a subject in a sequence and within a time interval
such that they provide an increased benefit than if they were
administered otherwise. Any additional therapy can be administered
in any order with the other additional therapy.
[0024] As used herein, the terms "manage," "managing," and
"management" in the context of the administration of a therapy to a
subject refer to the beneficial effects that a subject derives from
a therapy (e.g., a prophylactic or therapeutic vaccine) or a
combination of therapies, while not resulting in a cure of cancer.
In certain embodiments, a subject is administered one or more
therapies (e.g., one or more prophylactic or therapeutic vaccines)
to "manage" cancer so as to prevent the progression or worsening of
the condition.
[0025] As used herein, the terms "prevent," "preventing" and
"prevention" in the context of the administration of a therapy to a
subject refer to the prevention or inhibition of the recurrence,
onset, and/or development of brain cancer or a symptom thereof in a
subject resulting from the administration of a therapy (e.g., a
prophylactic or therapeutic agent), or a combination of therapies
(e.g., a combination of prophylactic or therapeutic agents).
[0026] As used herein, the term "concurrently" means sufficiently
close in time to produce a combined effect (that is, concurrently
may be simultaneously, or it may be two or more events occurring
within a time period before or after each other). When administered
with other agents, the EphaA2 and/or IL-13R.alpha.2 peptides
provided herein may be administered concurrently with the other
active agent. In some embodiments the EphaA2 and/or IL-13R.alpha.2
peptides provided herein and one or more other agents (e.g., a
helper T cell epitope, an adjuvant, and/or an immune response
modifier) are administered to a subject concurrently, wherein
administration of the EphaA2 and/or IL-13R.alpha.2 peptide and one
or more other agents are in the same composition. In other
embodiments an EphaA2 and/or IL-13R.alpha.2 peptide and one or more
other agents (e.g., a helper T cell epitope, an adjuvant, and/or an
immune response modifier) are administered to a subject
concurrently, wherein administration of the EphaA2 and/or
IL-13R.alpha.2 peptide and one or more other agents are not in the
same composition. In certain embodiments, an EphaA2 peptide and/or
IL-13R.alpha.2 provided herein and one or more other agents (e.g.,
a helper T cell epitope, an adjuvant, and/or an immune response
modifier) are administered to a subject concurrently, wherein the
concurrent administration is separated by at least 1 hour, 2 hours,
3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, 1 week, or 2 weeks.
[0027] As used herein, the term "EphA2 peptide" refers to a peptide
derived from the EphA2 protein. In a specific embodiment the EphA2
protein from which an EphA2 peptide is derived is the human EphA2
protein. In another specific embodiment, an EphA2 peptide comprises
or consists of the following amino acid sequence: TLADFDPRV (SEQ ID
NO:1). In some embodiments, an EphA2 peptide comprises one, two,
three, or more amino acid mutations (e.g., additions,
substitutions, or deletions) relative to the EphA2 peptide as it
exists in the native (e.g., wild-type) form of the EphA2
protein.
[0028] As used herein, the term "IL-13R.alpha.2 peptide" refers to
a peptide derived from the IL-13R.alpha.2 protein. In a specific
embodiment the IL-13R.alpha.2 protein from which an IL-13R.alpha.2
peptide is derived is the human IL-13R.alpha.2 protein. In another
specific embodiment, an IL-13R.alpha.2 peptide comprises or
consists of the following amino acid sequence: WLPFGFILI (SEQ ID
NO:2). In another specific embodiment, an IL-13R.alpha.2 peptide
comprises or consists of the following amino acid sequence:
WLPFGFILV (SEQ ID NO:3). In another specific embodiment, an
IL-13R.alpha.2 peptide comprises or consists of the following amino
acid sequence: ALPFGFILV (SEQ ID NO:4). In another specific
embodiment, an IL-13R.alpha.2 peptide comprises or consists of the
following amino acid sequence: ELPFGFILV (SEQ ID NO:5). In some
embodiments, an IL-13R.alpha.2 peptide comprises one, two, three,
or more amino acid mutations (e.g., additions, substitutions, or
deletions) relative to the IL-13R.alpha.2 peptide as it exists in
the native (e.g., wild-type) form of the IL-13R.alpha.2
protein.
[0029] As used herein and unless otherwise specified, the term
"antibody refers to a molecule with an antigen binding site that
immunospecifically binds an antigen. Antibodies include, but are
not limited to, monoclonal antibodies, polyclonal antibodies,
recombinantly produced antibodies, multispecific antibodies
(including bi-specific antibodies), human antibodies, humanized
antibodies, chimeric antibodies, synthetic antibodies, tetrameric
antibodies comprising two heavy chain and two light chain molecule,
an antibody light chain monomer, an antibody heavy chain monomer,
an antibody light chain dimer, an antibody heavy chain dimer, an
antibody light chain-antibody heavy chain pair, intrabodies,
heteroconjugate antibodies, single domain antibodies, monovalent
antibodies, single-chain Fvs (scFv) (e.g., including monospecific,
bispecific, etc.), camelized antibodies, Fab fragments, F(ab')
fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-anti-Id antibodies), and
epitope-binding fragments of any of the above. Antibodies can be of
any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g.,
IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a
or IgG2b) of immunoglobulin molecule. In certain embodiments,
antibodies described herein are IgG antibodies, or a class (e.g.,
human IgG1 or IgG4) or subclass thereof.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 demonstrates that the bulk of the cells of the A-172
cancer cell line express EphA2 and IL-13R.alpha.2 at high levels,
but only a fraction of these cells express CD133.
[0031] FIG. 2 depicts joint staining of CD133 and EphA2 cells of
the A-172 cancer cell line, and demonstrates that CD133+ cells of
the cell line also express EphA2.
[0032] FIG. 3 depicts joint staining of CD133 and IL-13R.alpha.2
cells of the A-172 cancer cell line, and demonstrates that CD133+
cells of the cell line also express IL-13R.alpha.2.
[0033] FIG. 4 shows that CD133+ cells of the A-172 cancer cell line
also express EphA2.
[0034] FIG. 5 shows that CD133+ cells of the A-172 cancer cell line
also express IL-13R.alpha.2.
[0035] FIG. 6 demonstrates that the bulk of the cells of the A-172
cancer cell line express EphA2 and IL-13R.alpha.2 at high levels,
but only a fraction of these cells express CD133.
[0036] FIG. 7 demonstrates that only a fraction of the cells of the
A-172 cancer cell line express CD133.
[0037] FIG. 8 demonstrates that CD133+ cells of the A-172 cancer
cell line also express EphA2 and IL-13R.alpha.2.
5. DETAILED DESCRIPTION
[0038] In one aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a peptide derived from the
EphA2 protein and monitoring the amount of cancer stem cells in
said subject. In a specific embodiment, the amount of cancer stem
cells in said subject that express EphA2 is measured. In another
specific embodiment, the amount of cancer stem cells in said
subject that express CD133 is measured. In another specific
embodiment, the amount of cancer stem cells in said subject that
express EphA2 and CD 133 is measured.
[0039] In another aspect, provided herein are methods for
monitoring the efficacy of an EphA2 peptide-based cancer treatment
(i.e., a treatment or therapy that comprises administration of a
peptide derived from EphA2) for a patient with cancer, comprising
monitoring the amount of cancer stem cells in said subject prior
to, during, and/or following the cancer treatment of a patient. In
a specific embodiment, the amount of cancer stem cells in said
subject that express EphA2 is measured. In another specific
embodiment, the amount of cancer stem cells in said subject that
express CD133 is measured. In another specific embodiment, the
amount of cancer stem cells in said subject that express EphA2 and
CD133 is measured.
[0040] In another aspect, provided herein are methods for treating
cancer comprising administering a T cell epitope of EphA2 that
targets cancer stem cells, wherein said epitope is sufficient to
induce an immune response in a patient with cancer.
[0041] In another aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a compound that targets the
EphA2 protein, wherein said compound is capable of killing and/or
preventing the differentiation of cancer stem cells that express
the EphA2 protein. In a specific embodiment, the compound is an
antibody that specifically binds EphA2.
[0042] In another aspect, provided herein are methods of improving
the targeting of cancer stem cells with a cancer vaccine comprising
determining the binding motif of a Class I or Class II epitope from
EphA2, and making substitutions in the amino acid sequence such
that the modified peptides are able to induce an immune response
that is at least as effective at killing cancer stem cells as the
wild type peptide.
[0043] In another aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a peptide derived from the
IL-13R.alpha.2 protein and monitoring the amount of cancer stem
cells in said subject. In a specific embodiment, the amount of
cancer stem cells in said subject that express IL-13R.alpha.2 is
measured. In another specific embodiment, the amount of cancer stem
cells in said subject that express CD133 is measured. In another
specific embodiment, the amount of cancer stem cells in said
subject that express IL-13R.alpha.2 and CD133 is measured.
[0044] In another aspect, provided herein are methods for
monitoring the efficacy of an IL-13R.alpha.2 peptide-based cancer
treatment (i.e., a treatment or therapy that comprises
administration of a peptide derived from IL-13R.alpha.2) for a
patient with cancer, comprising monitoring the amount of cancer
stem cells in said subject prior to, during, and/or following the
cancer treatment of a patient. In a specific embodiment, the amount
of cancer stem cells in said subject that express IL-13R.alpha.2 is
measured. In another specific embodiment, the amount of cancer stem
cells in said subject that express CD 133 is measured. In another
specific embodiment, the amount of cancer stem cells in said
subject that express IL-13R.alpha.2 and CD133 is measured.
[0045] In another aspect, provided herein are methods for treating
cancer comprising administering a T cell epitope of IL-13R.alpha.2
that targets cancer stem cells, wherein said epitope is sufficient
to induce an immune response in a patient with cancer.
[0046] In another aspect, provided herein are methods of treating
cancer in a subject, comprising administering to said subject a
therapeutically effective amount of a compound that targets the
IL-13R.alpha.2 protein, wherein said compound is capable of killing
and/or preventing the differentiation of cancer stein cells that
express the IL-13R.alpha.2 protein. In a specific embodiment, the
compound is an antibody that specifically binds IL-13R.alpha.2.
[0047] In another aspect, provided herein are methods of improving
the targeting of cancer stem cells with a cancer vaccine comprising
determining the binding motif of a Class I or Class II epitope from
IL-13R.alpha.2, and making substitutions in the amino acid sequence
such that the modified peptides are able to induce an immune
response that is at least as effective at killing cancer stem cells
as the wild type peptide.
5.1 Methods of Monitoring Cancer Stem Cells
[0048] As part of the prophylactically effective and/or
therapeutically effective regimens described herein, cancer stem
cells can be monitored to assess the efficacy of an EphA2 peptide
based cancer therapy or an IL-13R.alpha.2 peptide based cancer
therapy as well as to determine prognosis of a subject with cancer
or the efficacy of a therapeutically or prophylactically effective
regimen. In certain embodiments of the prophylactically effective
and/or therapeutically effective therapies or regimens described
herein, the therapies or regimens result in a stabilization or
reduction of cancer stem cells in the patient. In one embodiment,
the subject undergoing the regimen is monitored to assess whether
the regimen has resulted in a stabilization or reduction in the
cancer stem cells in the subject. In specific embodiments, the
methods of monitoring measure an EphA2-, IL-13R.alpha.2-, and/or
CD133-expressing cancer stem cells in the subjects to whom an an
EphA2 peptide based cancer therapy or an IL-13R.alpha.2 peptide
based cancer therapy is administered.
[0049] Without being limited by any particular theory or mechanism
of action, cancer stem cells, e.g., EphA2-expressing cancer stem
cells and/or IL-13R.alpha.2-expressing cancer stem cells, comprise
a unique subpopulation (e.g., 0.1-10% or so) of a tumor that, in
contrast to the remaining 90% or so of the tumor (i.e., the tumor
bulk), are relatively more tumorigenic and relatively more
slow-growing or quiescent. Given that conventional therapies and
regimens have, in large part, been designed to attack rapidly
proliferating cells (i.e., those cancer cells that comprise the
tumor bulk), slower growing cancer stem cells may be relatively
more resistant than faster growing tumor bulk to conventional
therapies and regimens. This would explain another reason for the
failure of standard oncology treatment regimens to ensure long-term
benefit in most patients with advanced stage cancers. In a specific
embodiment, an EphA2-expressing cancer stem cell or
IL-13R.alpha.2-expressing cancer stein cell is the founder cell of
a tumor (i.e., it is the progenitor of cancer cells). In some
embodiments, an EphA2-expressing cancer stem cell or
IL-13R.alpha.2-expressing cancer stem cell has one, two, three, or
more or all of the following characteristics or properties: (i) can
harbor the ability to initiate a tumor and/or to perpetuate tumor
growth, (ii) can be generally relatively less mutated than the bulk
of a tumor (e.g. due to slower growth and thus fewer DNA
replication-dependent errors, improved DNA repair, and/or
epigenetic/non-mutagenic changes contributing to their malignancy),
(iii) can have many features of a normal stem cell(s) (e.g.,
similar cell surface antigen and/or intracellular expression
profile, self-renewal programs, multi-drug resistance, an immature
phenotype, etc., characteristic of normal stein cells) and may be
derived from a normal stem cell(s), (iv) can be potentially
responsive to its microenvironment (e.g., the cancer stem cells may
be capable of being induced to differentiate and/or divide
asymmetrically), (v) can be the source of metastases, (vi) can be
slow-growing or quiescent, (vii) can be symmetrically-dividing,
(viii) can be tumorigenic (e.g. as determined by NOD/SCID
implantation experiments), (ix) can be relatively resistant to
traditional therapies (i.e. chemoresistant), and (x) can comprise a
subpopulation of a tumor (e.g. relative to the tumor bulk).
[0050] In certain embodiments, the amount of cancer stem cells in a
sample from a subject is determined/assessed using a technique
described herein or well-known to one of skill in the art. Such
samples include, but are not limited to, biological samples and
samples derived from a biological sample. In certain embodiments,
in addition to the biological sample itself or in addition to
material derived from the biological sample such as cells, the
sample used in the methods of this invention comprises added water,
salts, glycerin, glucose, an antimicrobial agent, paraffin, a
chemical stabilizing agent, heparin, an anticoagulant, or a
buffering agent. In certain embodiments, the biological sample is
blood, serum, urine, bone marrow or interstitial fluid. In another
embodiment, the sample is a tissue sample. In a particular
embodiment, the tissue sample is breast, brain, skin, colon, lung,
liver, ovarian, pancreatic, prostate, renal, bone or skin tissue.
In a specific embodiment, the tissue sample is a biopsy of normal
or tumor tissue. The amount of biological sample taken from the
subject will vary according to the type of biological sample and
the method of detection to be employed. In a particular embodiment,
the biological sample is blood, serum, urine, or bone marrow and
the amount of blood, serum, urine, or bone marrow taken from the
subject is 0.1 ml, 0.5 ml, 1 ml, 5 ml, 8 ml, 10 ml or more. In
another embodiment, the biological sample is a tissue and the
amount of tissue taken from the subject is less than 10 milligrams,
less than 25 milligrams, less than 50 milligrams, less than 1 gram,
less than 5 grams, less than 10 grams, less than 50 grams, or less
than 100 grams.
[0051] In accordance with the methods described herein, a sample
derived from a biological sample is one in which the biological
sample has been subjected to one or more pretreatment steps prior
to the detection and/or measurement of the cancer stem cell
population in the sample. In certain embodiments, a biological
fluid is pretreated by centrifugation, filtration, precipitation,
dialysis, or chromatography, or by a combination of such
pretreatment steps. In other embodiments, a tissue sample is
pretreated by freezing, chemical fixation, paraffin embedding,
dehydration, permeabilization, or homogenization followed by
centrifugation, filtration, precipitation, dialysis, or
chromatography, or by a combination of such pretreatment steps. In
certain embodiments, the sample is pretreated by removing cells
other than stem cells or cancer stem cells from the sample, or
removing debris from the sample prior to the determination of the
amount of cancer stem cells in the sample according to the methods
of the invention.
[0052] The samples for use in the methods of described herein may
be taken from any animal subject, preferably mammal, most
preferably a human. The subject from which a sample is obtained and
utilized in accordance with the methods of this invention includes,
without limitation, an asymptomatic subject, a subject manifesting
or exhibiting 1, 2, 3, 4 or more symptoms of cancer, a subject
clinically diagnosed as having cancer, a subject predisposed to
cancer, a subject suspected of having cancer, a subject undergoing
therapy for cancer, a subject that has been medically determined to
be free of cancer (e.g., following therapy for the cancer), a
subject that is managing cancer, or a subject that has not been
diagnosed with cancer. In certain embodiments, the term "has no
detectable cancer," as used herein, refers to a subject or subjects
in which there is no detectable cancer by conventional methods,
e.g., MRI. In other embodiments, the term refers to a subject or
subjects free from any disorder.
[0053] In certain embodiments, the amount of cancer stem cells in a
subject or a sample from a subject is/are assessed prior to therapy
or regimen (e.g. at baseline) or at least 1, 2, 4, 6, 7, 8, 10, 12,
14, 15, 16, 18, 20, 30, 60, 90 days, 6 months, 9 months, 12 months,
or >12 months after the subject begins receiving the therapy or
regimen. In certain embodiments, the amount of cancer stem cells is
assessed after a certain number of doses (e.g., after 2, 5, 10, 20,
30 or more doses of a therapy). In other embodiments, the amount of
cancer stein cells is assessed after 1 week, 2 weeks, 1 month, 2
months, 1 year, 2 years, 3 years, 4 years or more after receiving
one or more therapies.
[0054] In certain embodiments, a positive or negative control
sample is a sample that is obtained or derived from a corresponding
tissue or biological fluid or tumor as the sample to be analyzed in
accordance with the methods of the invention. This sample may come
from the same patient or different persons and at the same or
different time points.
[0055] For clarity of disclosure, and not by way of limitation, the
following pertains to analysis of a blood sample from a patient.
However, as one skilled in the art will appreciate, the assays and
techniques described herein can be applied to other types of
patient samples, including a body fluid (e.g. blood, bone marrow,
plasma, urine, bile, ascitic fluid), a tissue sample suspected of
containing material derived from a cancer (e.g. a biopsy) or
homogenate thereof. The amount of sample to be collected will vary
with the particular type of sample and method of determining the
amount of cancer stein cells used and will be an amount sufficient
to detect the cancer stein cells in the sample.
[0056] A sample of blood may be obtained from a patient having
different developmental or disease stages. Blood may be drawn from
a subject from any part of the body (e.g., a finger, a hand, a
wrist, an arm, a leg, a foot, an ankle, a stomach, and a neck)
using techniques known to one of skill in the art, in particular
methods of phlebotomy known in the art. In a specific embodiment,
venous blood is obtained from a subject and utilized in accordance
with the methods of the invention. In another embodiment, arterial
blood is obtained and utilized in accordance with the methods of
the invention. The composition of venous blood varies according to
the metabolic needs of the area of the body it is servicing. In
contrast, the composition of arterial blood is consistent
throughout the body. For routine blood tests, venous blood is
generally used.
[0057] The amount of blood collected will vary depending upon the
site of collection, the amount required for a method of the
invention, and the comfort of the subject. In some embodiments, any
amount of blood is collected that is sufficient to detect the
amount of cancer stem cells. In a specific embodiment, 1cc or more
of blood is collected from a subject.
[0058] The amount of cancer stem cells in a sample can be expressed
as the percentage of, e.g., overall cells, overall cancer cells or
overall stem cells in the sample, or quantitated relative to area
(e.g. cells per high power field), or volume (e.g. cells per ml),
or architecture (e.g. cells per bone spicule in a bone marrow
specimen).
[0059] In some embodiments, the sample may be a blood sample, bone
marrow sample, or a tissue/tumor biopsy sample, wherein the amount
of cancer stem cells per unit of volume (e.g., 1 mL) or other
measured unit (e.g., per unit field in the case of a histological
analysis) is quantitated. In certain embodiments, the cancer stem
cell population is determined as a portion (e.g., a percentage) of
the cancerous cells present in the blood or bone marrow or
tissue/tumor biopsy sample or as a subset of the cancerous cells
present in the blood or bone marrow or tissue/tumor biopsy sample.
The cancer stem cell population, in other embodiments, can be
determined as a portion (e.g., percentage) of the total cells. In
yet other embodiments, the cancer stem cell population is
determined as a portion (e.g., a percentage) of the total stem
cells present in the blood sample.
[0060] In other embodiments, the sample from the patient is a
tissue sample (e.g., a biopsy from a subject with or suspected of
having cancerous tissue), where the amount of cancer stem cells can
be measured, for example, by immunohistochemistry or flow
cytometry, or on the basis of the amount of cancer stem cells per
unit area, volume, or weight of the tissue. In certain embodiments,
the cancer stem cell population (the amount of cancer stem cells)
is determined as a portion (e.g., a percentage) of the cancerous
cells present in the tissue sample or as a subset of the cancerous
cells present in the tissue sample. In yet other embodiments, the
cancer stem cell population is determined as a portion (e.g., a
percentage) of the overall cells or stem cell cells in the tissue
sample.
[0061] The amount of cancer stem cells in a test sample can be
compared with the amount of cancer stem cells in reference
sample(s) to assess the efficacy of the regimen. In one embodiment,
the reference sample is a sample obtained from the subject
undergoing therapy at an earlier time point (e.g., prior to
receiving the regimen as a baseline reference sample, or at an
earlier time point while receiving the therapy). In this
embodiment, the therapy desirably results in a decrease in the
amount of cancer stem cells in the test sample as compared with the
reference sample. In another embodiment, the reference sample is
obtained from a healthy subject who has no detectable cancer, or
from a patient that is in remission for the same type of cancer. In
this embodiment, the therapy desirably results in the test sample
having an equal amount of cancer stem cells, or less than the
amount of cancer stem cells than are detected in the reference
sample.
[0062] In other embodiments, the cancer stem cell population in a
test sample can be compared with a predetermined reference range
and/or a previously detected amount of cancer stem cells determined
for the subject to gauge the subject's response to the regimens
described herein. In a specific embodiment, a stabilization or
reduction in the amount of cancer stem cells relative to a
predetermined reference range and/or earlier (previously detected)
cancer stem cell amount determined for the subject indicates an
improvement in the subject's prognosis or a positive response to
the regimen, whereas an increase relative to the predetermined
reference range and/or earlier cancer stem cell amount indicates
the same or worse prognosis, and/or a failure to respond to the
regimen. The cancer stem cell amount can be used in conjunction
with other measures to assess the prognosis of the subject and/or
the efficacy of the regimen. In a specific embodiment, the
predetermined reference range is based on the amount of cancer stem
cells obtained from a patient or population(s) of patients
suffering from the same type of cancer as the patient undergoing
the therapy.
[0063] Generally, since stem cell antigens, e.g., EphA2, CD133, and
IL-13R.alpha.2, can be present on both cancer stem cells and normal
stem cells, a sample from the cancer-afflicted patient will have a
higher stein cell count than a sample from a healthy subject who
has no detectable cancer, due to the presence of the cancer stem
cells. The therapy will desirably result in a cancer stem cell
count for the test sample (e.g., the sample from the patient
undergoing therapy) that decreases and becomes increasingly closer
to the stein cell count in a reference sample that is sample from a
healthy subject who has no detectable cancer.
[0064] If the reduction in amount of cancer stem cells is
determined to be inadequate upon comparing the amount of cancer
stem cells in the sample from the subject undergoing the regimen
with the reference sample, then the medical practitioner has a
number of possible options to adjust the regimen. For instance, the
medical practitioner can then increase either the dosage or
intensity of the therapy administered, the frequency of the
administration, the duration of administration, combine the therapy
with another therapy(ies), change the management altogether
including halting therapy, or any combination thereof.
[0065] In certain embodiments, the dosage, frequency and/or
duration of administration of a therapy is modified as a result of
the change in the amount of cancer stein cells detected in or from
the treated patient. For example, if a subject receiving therapy
for leukemia has an cancer stem cell measurement of 2.5% of his
tumor prior to therapy and 5% after 6 weeks of therapy, then the
therapy or regimen may be altered or stopped because the increase
in the percentage of cancer stem cells indicates that the therapy
or regimen is not optimal. Alternatively, if another subject has an
cancer stein cell measurement of 2.5% of his tumor prior to therapy
and 1% after 6 weeks of therapy, then the therapy or regimen may be
continued because the decrease in the percentage of cancer stein
cells indicates that the therapy or regimen is effective.
[0066] The amount of cancer stem cells can be monitored/assessed
using standard techniques known to one of skill in the art. Cancer
stem cells can be monitored by, e.g., obtaining a sample, such as a
tissue/tumor sample, blood sample or a bone marrow sample, from a
subject and detecting cancer stem cells in the sample. The amount
of cancer stem cells in a sample (which may be expressed as
percentages of, e.g., overall cells or overall cancer cells) can be
assessed by detecting the expression cancer stem cell antigens
(e.g., EphA2) on cancer stem cells. Techniques known to those
skilled in the art can be used for measuring these activities.
Antigen expression can be assayed, for example, by immunoassays
including, but not limited to, western blots, immunohistochemistry,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays,
immunofluorescence, protein A immunoassays, flow cytometry, and
FACS analysis. In such circumstances, the amount of cancer stem
cells in a test sample from a subject may be determined by
comparing the results to the amount of cancer stem cells in a
reference sample (e.g., a sample from a subject who has no
detectable cancer) or to a predetermined reference range, or to the
patient him/herself at an earlier time point (e.g. prior to, or
during therapy).
[0067] In a specific embodiment, the cancer stem cell population in
a sample from a patient is determined by flow cytometry. This
method exploits the differential expression of certain surface
markers on cells. Labeled antibodies (e.g., fluorescent antibodies)
specific to cancer stem cells antigens (e.g., EphA2) can be used to
react with the cells in the sample, and the cells are subsequently
sorted by FACS methods. In some embodiments, a combination of cell
surface markers are utilized in order to determine the amount of
cancer stem cells in the sample. For example, both positive and
negative cell sorting may be used to assess the amount of cancer
stem cells in the sample. In a specific embodiment the cancer stem
cell population in a sample, e.g., a tissue sample, such as a solid
tumor biopsy, is determined using immunohistochemistry techniques.
This method exploits the differential expression of certain surface
markers on cells. Labeled antibodies (e.g., fluorescent antibodies)
specific to cancer stem cells antigens (e.g., EphA2) can be used to
react with the cells in the sample, and the tissue is subsequently
stained. In some embodiments, a combination of certain cell surface
markers are utilized in order to determine the amount of cancer
stem cells in the sample.
[0068] In other embodiments, sphere formation can be used to
determine the amount of cancer stem cells in a sample (See Singh et
al., "Identification of a Cancer Stem Cell from Human Brain
Tumors," Cancer Res 63: 5821-5828 (2003).
[0069] In other embodiments, a sample (e.g., a tumor or normal
tissue sample, blood sample or bone marrow sample) obtained from
the patient is analyzed in in vivo systems to determine the cancer
stem cell population or amount of cancer stem cells. In certain
embodiments, for example, in vivo engraftment is used to quantitate
the amount of cancer stem cells in a sample. In vivo engraftment
involves implantation of a human specimen with the readout being
the formation of tumors in an animal such as in immunocompromised
or immunodeficient mice (such as NOD/SCID mice). Typically, the
patient sample is cultured or manipulated in vitro and then
injected into the mice. In these assays, mice can be injected with
a decreasing amount of cells from patient samples, and the
frequency of tumor formation can be plotted vs. the amount of cells
injected to determine the amount of cancer stem cells in the
sample. Alternatively, the rate of growth of the resulting tumor
can be measured, with larger or more rapidly advancing tumors
indicating a higher cancer stem cells amount in the patient sample.
In this way, an in vivo engraftment model/assay could be used to
measure cancer stem cells amount pre- and post-therapy to assess
the change in cancer stem cell amount arising from a given therapy
or regimen.
[0070] In certain in vivo techniques, an imaging agent or
diagnostic agent is used which binds to biological molecules on
cancer cells or cancer stem cells, e.g., binds to EphA2 on cancer
stem cells. For instance, a fluorescent tag, radionuclide, heavy
metal, or photon-emitter is attached to an antibody (including an
antibody fragment) that binds to EphA2. The medical practitioner
can infuse the labeled antibody into the patient either prior to,
during, or following treatment, and then the practitioner can place
the patient into a total body scanner/developer which can detect
the attached label (e.g., fluorescent tag, radionuclide, heavy
metal, photon-emitter). The scanner/developer (e.g., CT, MRI, or
other scanner, e.g. detector of fluorescent label, that can detect
the label) records the presence, amount/quantity, and bodily
location of the bound antibody. In this manner, the mapping and
quantitation of tag (e.g. fluorescence, radioactivity, etc.) in
patterns (i.e., different from patterns of normal stem cells within
a tissue) within a tissue or tissues indicates the treatment
efficacy within the patient's body when compared to a reference
control such as the same patient at an earlier time point or a
patient or healthy individual who has no detectable cancer. For
example, a large signal (relative to a reference range or a prior
treatment date, or prior to treatment) at a particular location
indicates the presence of cancer stem cells. If this signal is
increased relative to a prior date it suggests a worsening of the
disease and failure of therapy or regimen. Alternatively, a signal
decrease indicates that the therapy or regimen has been
effective.
[0071] In a specific embodiment, the amount of cancer stem cells is
detected in vivo in a subject according to a method comprising the
steps of: (a) administering to the subject an effective amount of a
labeled binding agent that specifically binds to an antigen of
cancer stem cells (e.g., EphA2 or CD133), and (b) detecting the
labeled agent in the subject following a time interval sufficient
to allow the labeled agent to concentrate at sites in the subject
where the cancer stem cell surface marker is expressed. In
accordance with this embodiment, the binding agent is administered
to the subject according to any suitable method in the art, for
example, parenterally (such as intravenously), or
intraperitoneally. In accordance with this embodiment, the
effective amount of the agent is the amount which permits the
detection of the agent in the subject. This amount will vary
according to the particular subject, the label used, and the
detection method employed. For example, it is understood in the art
that the size of the subject and the imaging system used will
determine the amount of labeled agent needed to detect the agent in
a subject using an imaging means. In the case of a radiolabeled
agent for a human subject, the amount of labeled agent administered
is measured in terms of radioactivity, for example from about 5 to
20 millicuries of 99Tc. The time interval following the
administration of the labeled agent which is sufficient to allow
the labeled agent to concentrate at sites in the subject where the
cancer stem cell surface marker is expressed will vary depending on
several factors, for example, the type of label used, the mode of
administration, and the part of the subject's body that is imaged.
In a particular embodiment, the time interval that is sufficient is
6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. In another
embodiment the time interval is 5 to 20 days or 5 to 10 days. The
presence of the labeled cancer stem cell surface marker-binding
agent can be detected in the subject using imaging means known in
the art. In general, the imaging means employed depend upon the
type of label used. Skilled artisans will be able to determine the
appropriate means for detecting a particular label. Methods and
devices that may be used include, but are not limited to, computed
tomography (CT), whole body scan such as position emission
tomography (PET), magnetic resonance imaging (MRI), an imager which
can detect and localize fluorescent label, and sonography. In a
specific embodiment, the cancer binding agent is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the binding agent is labeled
with a fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the binding agent is labeled with a positron emitting metal and is
detected in the patient using positron emission-tomography. In yet
another embodiment, the binding agent is labeled with a
paramagnetic label and is detected in a patient using magnetic
resonance imaging (MRI).
[0072] Any in vitro or in vivo (ex vivo) assays known to those
skilled in the art that can detect and/or quantify cancer stem
cells can be used to monitor cancer stem cells in or from a subject
in order to evaluate the prophylactic and/or therapeutic utility of
a cancer therapy or regimen disclosed herein for cancer or one or
more symptoms thereof; or these assays can be used to assess the
prognosis of a patient. The results of these assays then may be
used to possibly maintain or alter the cancer therapy or
regimen.
[0073] The amount of cancer stem cells in a specimen can be
compared to a predetermined reference range and/or an earlier
amount of cancer stem cells previously determined for the subject
(either prior to, or during therapy) in order to gauge the
subject's response to the treatment regimens described herein. In a
specific embodiment, a stabilization or reduction in the amount of
cancer stem cells relative to a predetermined reference range
and/or earlier cancer stem cell amount previously determined for
the subject (either prior to, or during therapy) indicates that the
therapy or regimen was effective and thus possibly an improvement
in the subject's prognosis, whereas an increase relative to the
predetermined reference range and/or cancer stem cell amount
detected at an earlier time point indicates that the therapy or
regimen was ineffective and thus possibly the same or a worsening
in the subject's prognosis. The cancer stem cell amount can be used
with other standard measures of cancer to assess the prognosis of
the subject and/or efficacy of the therapy or regimen: such as
response rate, durability of response, relapse-free survival,
disease-free survival, progression-free survival, and overall
survival. In certain embodiments, the dosage, frequency and/or
duration of administration of a therapy is modified as a result of
the determination of the amount or change in the amount of cancer
stem cells at various time points which may include prior to,
during, and/or following therapy.
[0074] Also provided herein are methods for determining that a
cancer therapy or regimen is effective at targeting and/or
impairing cancer stem cells by virtue of monitoring cancer stem
cells over time and detecting a stabilization or decrease in the
amount of cancer stem cells during and/or following the course of
the cancer therapy or regimen.
[0075] In a certain embodiment, a therapy or regimen may be
described or marketed as an anti-cancer stem cell therapy or
regimen based on the determination that a therapy or regimen is
effective at targeting and/or impairing cancer stem cells by virtue
of having monitored or detected a stabilization or decrease in the
amount of cancer stem cells during therapy.
[0076] Also provided herein are methods to treat cancer involving
i) determining that an EphA2-based cancer therapy is effective by
virtue of its ability to decrease cancer stein cells as determined
by the monitoring of cancer stem cells, and ii) administering the
therapy to a human(s) with cancer. Also provided herein are methods
to methods to treat cancer involving i) administering to a human
with cancer an EphA2-based cancer therapy, ii) determining the
amount of cancer stem cells prior to, during, and/or following
therapy through the monitoring of cancer stem cells, and iii)
continuing, altering, or halting therapy based on such monitoring.
Also provided herein are methods for assaying/screening of an
EphA2-based therapy(s) for anti-cancer stem cell activity involving
i) administration of the therapy to a human with cancer, ii)
monitoring cancer stem cells in or from the human prior to, during,
and/or following therapy, and iii) determining whether the therapy
resulted in a decrease in the amount of cancer stem cells.
[0077] Also provided herein are methods to treat cancer involving
i) determining that an IL-13R.alpha.2-based cancer therapy is
effective by virtue of its ability to decrease cancer stem cells as
determined by the monitoring of cancer stem cells, and ii)
administering the therapy to a human(s) with cancer. Also provided
herein are methods to methods to treat cancer involving i)
administering to a human with cancer an IL-13R.alpha.2-based cancer
therapy, ii) determining the amount of cancer stem cells prior to,
during, and/or following therapy through the monitoring of cancer
stem cells, and iii) continuing, altering, or halting therapy based
on such monitoring. Also provided herein are methods for
assaying/screening of an IL-13R.alpha.2-based therapy(s) for
anti-cancer stein cell activity involving i) administration of the
therapy to a human with cancer, ii) monitoring cancer stem cells in
or from the human prior to, during, and/or following therapy, and
iii) determining whether the therapy resulted in a decrease in the
amount of cancer stem cells.
5.2 Types of Cancer
[0078] With any type of cancer for which a patient can be treated,
the cancer stem cells thereof can be monitored in accordance with
the methods described herein. The medical practitioner can diagnose
the patient using any of the conventional cancer screening methods
including, but not limited to physical examination (e.g., prostate
examination, rectal examination, breast examination, lymph nodes
examination, abdominal examination, skin surveillance, testicular
exam, general palpation), visual methods (e.g., colonoscopy,
bronchoscopy, endoscopy), PAP smear analyses (cervical cancer),
stool guaiac analyses, blood tests (e.g., complete blood count
(CBC) test, prostate specific antigen (PSA) test, carcinoembryonic
antigen (CEA) test, cancer antigen (CA)-125 test, alpha-fetoprotein
(AFP), liver function tests), karyotyping analyses, bone marrow
analyses (e.g., in cases of hematological malignancies), histology,
cytology, flow cytometry, a sputum analysis and imaging methods
(e.g., computed tomography (CT), magnetic resonance imaging (MRI),
ultrasound, X-ray imaging, mammography, PET scans, bone scans).
[0079] Non-limiting examples of cancers include: leukemias, such as
but not limited to, acute leukemia, acute lymphocytic leukemia,
acute myelocytic leukemias, such as, myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia leukemias and
myelodysplastic syndrome (MDS); chronic leukemias, such as but not
limited to, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, hairy cell leukemia; polycythemia vera;
lymphomas such as but not limited to Hodgkin's disease,
non-Hodgkin's disease; multiple myelomas such as but not limited to
smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic
myeloma, plasma cell leukemia, solitary plasmacytoma and
extramedullary plasmacytoma; Waldenstrom's macroglobulinemia;
monoclonal gammopathy of undetermined significance; benign
monoclonal gammopathy; heavy chain disease; bone and connective
tissue sarcomas such as but not limited to bone sarcoma,
osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell
tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma,
soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma,
Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,
neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such
as but not limited to, astrocytoma, brain stem glioma, ependymoma,
oligodendroglioma, nonglial tumor, acoustic neurinoma,
craniopharyngioma, medulloblastoma, meningioma, pineocytoma,
pineoblastoma, primary brain lymphoma; breast cancer including but
not limited to ductal carcinoma, adenocarcinoma, lobular (small
cell) carcinoma, intraductal carcinoma, medullary breast cancer,
mucinous breast cancer, tubular breast cancer, papillary breast
cancer, Paget's disease, and inflammatory breast cancer; adrenal
cancer such as but not limited to pheochromocytom and
adrenocortical carcinoma; thyroid cancer such as but not limited to
papillary or follicular thyroid cancer, medullary thyroid cancer
and anaplastic thyroid cancer; pancreatic cancer such as but not
limited to, insulinoma, gastrinoma, glucagonoma, vipoma,
somatostatin-secreting tumor, and carcinoid or islet cell tumor;
pituitary cancers such as but limited to Cushing's disease,
prolactin-secreting tumor, acromegaly, and diabetes insipius; eye
cancers such as but not limited to ocular melanoma such as iris
melanoma, choroidal melanoma, and cilliary body melanoma, and
retinoblastoma; vaginal cancers such as squamous cell carcinoma,
adenocarcinoma, and melanoma; vulvar cancer such as squamous cell
carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,
and Paget's disease; cervical cancers such as but not limited to,
squamous cell carcinoma, and adenocarcinoma; uterine cancers such
as but not limited to endometrial carcinoma and uterine sarcoma;
ovarian cancers such as but not limited to, ovarian epithelial
carcinoma, borderline tumor, germ cell tumor, and stromal tumor;
esophageal cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers
such as but not limited to, adenocarcinoma, fungating (polypoid),
ulcerating, superficial spreading, diffusely spreading, malignant
lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon
cancers; rectal cancers; liver cancers such as but not limited to
hepatocellular carcinoma and hepatoblastoma; gallbladder cancers
such as adenocarcinoma; cholangiocarcinomas such as but not limited
to papillary, nodular, and diffuse; lung cancers such as non-small
cell lung cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers such as but not limited to germinal tumor,
seminoma, anaplastic, classic (typical), spermatocytic,
nonseminoma, embryonal carcinoma, teratoma carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers such as but not
limited to, prostatic intraepithelial neoplasia, adenocarcinoma,
leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers
such as but not limited to squamous cell carcinoma; basal cancers;
salivary gland cancers such as but not limited to adenocarcinoma,
mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx
cancers such as but not limited to squamous cell cancer, and
verrucous; skin cancers such as but not limited to, basal cell
carcinoma, squamous cell carcinoma and melanoma, superficial
spreading melanoma, nodular melanoma, lentigo malignant melanoma,
acral lentiginous melanoma; kidney cancers such as but not limited
to renal cell carcinoma, adenocarcinoma, hypernephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/or
uterer); Wilms' tumor; bladder cancers such as but not limited to
transitional cell carcinoma, squamous cell cancer, adenocarcinoma,
carcinosarcoma. In addition, cancers include myxosarcoma,
osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma,
mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma,
cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma and papillary
adenocarcinomas (for a review of such disorders, see Fishman et
al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and
Murphy et al., 1997, Informed Decisions: The Complete Book of
Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin
Books U.S.A., Inc., United States of America).
[0080] Other cancers or other abnormal proliferative diseases,
include but are not limited to, the following: carcinoma, including
that of the bladder, breast, colon, kidney, liver, lung, ovary,
pancreas, stomach, cervix, thyroid and skin; including squamous
cell carcinoma; hematopoietic tumors of lymphoid lineage, including
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T cell lymphoma, Burkitt's lymphoma; hematopoietic
tumors of myeloid lineage, including acute and chronic myelogenous
leukemias and promyelocytic leukemia; tumors of mesenchymal origin,
including fibrosarcoma and rhabdomyoscarcoma; other tumors,
including melanoma, seminoma, tetratocarcinoma, neuroblastoma and
glioma; tumors of the central and peripheral nervous system,
including astrocytoma, neuroblastoma, glioma, and schwannomas;
tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including
melanoma, xeroderma pigmentosum, keratoactanthoma, seminoma,
thyroid follicular cancer and teratocarcinoma. Cancers associated
with aberrations in apoptosis are also included and are not be
limited to, follicular lymphomas, carcinomas with p53 mutations,
hormone dependent tumors of the breast, prostate and ovary, and
precancerous lesions such as familial adenomatous polyposis, and
myelodysplastic syndromes. In specific embodiments, malignancy or
dysproliferative changes (such as metaplasias and dysplasias), or
hyperproliferative disorders of the skin, lung, liver, bone, brain,
stomach, colon, breast, prostate, bladder, kidney, pancreas, ovary,
and/or uterus are encompassed in the invention.
[0081] Non-limiting examples of leukemias and other blood-borne
cancers include acute lymphoblastic leukemia "ALL", acute
lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,
acute myeloblastic leukemia "AML", acute promyelocytic leukemia
"APL", acute monoblastic leukemia, acute erythroleukemic leukemia,
acute megakaryoblastic leukemia, acute myelomonocytic leukemia,
acute nonlymphocyctic leukemia, acute undifferentiated leukemia,
chronic myelocytic leukemia "CML", chronic lymphocytic leukemia
"CLL", and hairy cell leukemia.
[0082] Non-limiting examples of lymphomas include Hodgkin's
disease, non-Hodgkin's Lymphoma, Multiple myeloma, Waldenstrom's
macroglobulinemia, Heavy chain disease, and Polycythemia vera.
[0083] Non-limiting examples of solid tumors encompassed in the
invention include, but are not limited to fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal
cancer, kidney cancer, pancreatic cancer, bone cancer, breast
cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach
cancer, oral cancer, nasal cancer, throat cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, uterine cancer, testicular cancer, small
cell lung carcinoma, bladder carcinoma, lung cancer, epithelial
carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, and retinoblastoma.
5.2.1 Brain Cancers
[0084] In a specific embodiment, the methods described herein can
be used in the prevention, treatment, and/or management of brain
cancer. In certain embodiments, such methods comprise a step of
monitoring the levels of cancer stem cells in a subject with brain
cancer that has been treated in accordance with the methods
described herein, e.g., the subject has been administered an
EphA2-based cancer therapy, i.e., an EphA2 peptide or a compound
(e.g., an antibody) that specifically targets EphA2 (e.g., targets
EphA2 present on EphA2-expressing cancer stem cells).
[0085] In another specific embodiment, the methods described herein
can be used in the prevention, treatment, and/or management of
brain cancer. In certain embodiments, such methods comprise a step
of monitoring the levels of cancer stem cells in a subject with
brain cancer that has been treated in accordance with the methods
described herein, e.g., the subject has been administered an
IL-13R.alpha.2-based cancer therapy, i.e., an IL-13R.alpha.2
peptide or a compound (e.g., an antibody) that specifically targets
IL-13R.alpha.2 (e.g., targets IL-13R.alpha.2 present on
IL-13R.alpha.2-expressing cancer stem cells).
[0086] Any type of brain cancer can be treated in accordance with
the methods described herein. Exemplary brain cancers include, but
are not limited to, gliomas (including astrocytoma (e.g., pilocytic
astrocytoma, diffuse astrocytoma, and anaplastic astrocytoma),
glioblastoma, oligodendroglioma, brain stem glioma, non-brain stem
glioma, ependymoma, and mixed tumors comprising more than one glial
cell types), acoustic schwannoma, cranialpharyngioma, meningioma,
medulloblastoma, primary central nervous system lymphoma, and
tumors of the pineal (e.g., pineal astrocytic tumors and pineal
parenchymal tumors) and pituitary glands. Gliomas additionally
include recurrent malignant gliomas, high-risk WHO Grade II
Astrocytomas, Oligo Astrocytomas, recurrent WHO Grade II Gliomas,
newly-diagnosed malignant or intrinsic brain stem gliomas,
incompletely resected non-brainstem gliomas, and recurrent
unresectable low-grade gliomas. Additional types of brain cancer
that can be treated in accordance with the methods described herein
include adult low-grade infiltrative supratentorial
astrocytoma/oligodendroglioma, adult low-grade infiltrative
supratentorial astrocytoma, adult low-grade infiltrative
supratentorial oligodendroglioma, adult low-grade infiltrative
supratentorial astrocytoma/oligodendroglioma (excluding pilocytic
astrocytoma), adult low-grade infiltrative supratentorial
astrocytoma (excluding pilocytic astrocytoma), adult low-grade
infiltrative supratentorial oligodendroglioma (excluding pilocytic
astrocytoma), adult intracranial ependymoma, adult intracranial
ependymoma (excluding subependymoma and myxopapillary), adult
intracranial anaplastic ependymoma, anaplastic glioma, anaplastic
glioblastoma, pilocytic astrocytoma, subependymoma, myxopapillary,
1 to 3 limited metastatic lesions (intraparenchymal), greater than
3 metastatic lesions (intraparenchymal), leptomeningeal metastases
(neoplastic meningitis), primary CNS lymphoma, metastatic spine
tumors, or meningiomas.
[0087] In one embodiment, the brain cancer treated in accordance
with the methods described herein is a glioma. In a specific
embodiment, the brain cancer treated in accordance with the methods
described herein is recurrent malignant glioma. In another specific
embodiment, the brain cancer treated in accordance with the methods
described herein is recurrent WHO Grade II Glioma. In another
specific embodiment, the brain cancer treated in accordance with
the methods described herein is newly-diagnosed malignant or
intrinsic brain stem glioma. In another specific embodiment, the
brain cancer treated in accordance with the methods described
herein is incompletely resected non-brainstem glioma. In another
specific embodiment, the brain cancer treated in accordance with
the methods described herein is recurrent unresectable low-grade
glioma.
[0088] In one embodiment, a patient treated in accordance with the
methods described herein is an adult with recurrent malignant
glioma, recurrent glioblastoma, anaplastic astrocytoma, anaplastic
oligodendroglioma, or anaplastic mixed oligoastrocytoma. In another
embodiment, the patient is an adult with newly diagnosed high-risk
low grade glioma. In another embodiment, the patient is an adult
with newly diagnosed high-risk low grade astrocytoma. In another
embodiment, the patient is an adult with newly diagnosed high-risk
low grade oligoastrocytoma. In another embodiment, the patient is
an adult with recurrent high-risk low grade astrocytoma. In another
embodiment, the patient is an adult with recurrent high-risk low
grade oligoastrocytoma. In another embodiment, the patient is an
adult with recurrent high-risk low grade oligodendroglioma. In
another embodiment, the patient is a child with newly diagnosed
malignant glioma. In another embodiment, the patient is a child
with intrinsic brain stem glioma. In another embodiment, the
patient is a child with incompletely resected non-brainsteam
high-grade glioma. In another embodiment, the patient is a child
with recurrent unresectable low-grade glioma. In another
embodiment, the patient is a child with newly diagnosed diffuse
intrinsic pontine glioma. In another embodiment, the patient is a
child with any high-grade glioma involving the brainstem and
treated with RT or without chemotherapy during RT. In another
embodiment, the patient is a child with newly diagnosed
non-brainstem high-grade glioma treated with RT with chemotherapy.
In another embodiment, the patient is a child with newly diagnosed
non-brainstem high-grade glioma treated with RT without
chemotherapy. In another embodiment, the patient is a child with
recurrent non-brainstem high-grade glioma that has recurred after
treatment.
[0089] In another embodiment, the brain cancer treated in
accordance with the methods described herein is an astrocytoma. In
a specific embodiment, the brain cancer treated in accordance with
the methods described herein is high-risk WHO Grade II Astrocytoma.
In another specific embodiment, the brain cancer treated in
accordance with the methods described herein is Oligo
Astrocytoma.
5.3 Peptides
5.3.1 Peptides Derived from EphA2
[0090] EphA2 is a tyrosine kinase receptor that is involved in the
formation of the notochord via interaction with ephrinA1. (see,
e.g., Naruse-Nakajima et al., Mech. Dev., 102: 95-105, 2001).
[0091] Any EphA2 peptide capable of serving as an HLA-A2 restricted
cytotoxic T lymphocyte (CTL) epitope may be used in accordance with
the described herein. In some embodiments, the EphA2 peptide used
in a vaccine described herein comprises SEQ ID NO:1. In some
embodiments, the EphA2 peptide used in a vaccine described herein
consists of SEQ ID NO:1.
[0092] In some embodiments, the EphA2 peptide used in accordance
with the methods described herein comprises a mutated version of an
EphA2 peptide, e.g., a mutated version of SEQ ID NO:1, wherein the
mutated version comprises at least 1, at least 2, or at least 3
amino acid substitutions (e.g., conservative substitutions),
additions, or deletions.
[0093] In some embodiments, the EphA2 peptide used in accordance
with the methods described herein comprises an amino acid sequence
with at least 50%, 60%, 70%, 80%, or 90% identity to SEQ ID NO:1.
In other embodiments, the EphA2 peptide used in accordance with the
methods described herein comprises an amino acid sequence with at
least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%,
or 80% to 90% identity to SEQ ID NO:1. In some embodiments, the
EphA2 peptide used in accordance with the methods described herein
comprises an amino acid sequence with at least 50%, 60%, 70%, 80%,
or 90% similarity to SEQ ID NO:1. In other embodiments, the EphA2
peptide used in accordance with the methods described herein
comprises an amino acid sequence with at least 50% to 60%, 50% to
70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% similarity
to SEQ ID NO:1. In specific embodiments, the EphA2 peptide used in
accordance with the methods described herein does not comprise or
consist of SEQ ID NO:1, i.e., the EphA2 peptide is derived from a
different portion of EphA2 than is SEQ ID NO:1.
5.3.2 Peptides Derived from IL-13R.alpha.2
[0094] IL-13R.alpha.2 a membrane glycoprotein that binds as a
component of a heterodimer to the Th2 cytokine, IL-13, which
induces monocytes and macrophages to produce TGF.beta. (see, e.g.,
Fichtner-Feigl et al., Nat. Med., 12: 99-106, 2006).
[0095] Any IL-13R.alpha.2 peptide capable of serving as an HLA-A2
restricted cytotoxic T lymphocyte (CTL) epitope may be used in a
vaccine described herein. In some embodiments, the IL-13R.alpha.2
peptide used in accordance with the methods described herein
comprises any one of SEQ ID NOs:2-5.
[0096] In some embodiments, the IL-13R.alpha.2 peptide used in a
vaccine described herein comprises a mutated version of SEQ ID
NO:2, wherein the mutated version of SEQ ID NO:2 comprises at least
1, at least 2, or at least 3 amino acid substitutions (e.g.,
conservative substitutions), additions, or deletions.
[0097] In some embodiments, the IL-13R.alpha.2 peptide used in a
vaccine described herein comprises an amino acid sequence with at
least 50%, 60%, 70%, 80%, or 90% identity to SEQ ID NO:2. In other
embodiments, the IL-13R.alpha.2 peptide used in a vaccine described
herein comprises an amino acid sequence with at least 50% to 60%,
50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90%
identity to SEQ ID NO:2. In some embodiments, the IL-13R.alpha.2
peptide used in a vaccine described herein comprises an amino acid
sequence with at least 50%, 60%, 70%, 80%, or 90% similarity to SEQ
ID NO:2. In other embodiments, the IL-13R.alpha.2 peptide used in a
vaccine described herein comprises an amino acid sequence with at
least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%,
or 80% to 90% similarity to SEQ ID NO:2.
5.4 Immune Response Modifiers
[0098] In some embodiments, the EphA2 and/or IL-13R.alpha.2
peptides provided herein and compositions thereof are administered
concurrently with an immune response modifier. Immune response
modifiers include agents capable of modifying the immune response
of a subject. In some embodiments, an immune response modifier
polarizes the immune response of a subject toward a Th1 response.
In other embodiments, an immune response modifier polarizes the
immune response of a subject toward a Th2 response. In a specific
embodiment, the immune response modifier binds to a toll-like
receptor (TLR) such as TLR3. Exemplary immune response modifiers
that can be administered concurrently with the EphA2 and/or
IL-13R.alpha.2 peptides provided herein include, without
limitation, Polyinosinic-Polycytidylic acid stabilized with
polylysine and carboxymethylcellulose (poly-ICLC; also known as
Hiltonol), imiquimod (Aldara.RTM.; Beselna.RTM.), and MIS-416
(Innate Therapeutics).
5.5 Adjuvants
[0099] In some embodiments, the EphA2 and/or IL-13R.alpha.2
peptides provided herein are administered concurrently with an
adjuvant. In some embodiments, the term "adjuvant" refers to an
agent that when administered concurrently with or in the same
composition as an EphA2 and/or IL-13R.alpha.2 peptide augments,
accelerates, prolongs, enhances and/or boosts the immune response
to the ILEphA2 and/or IL-13R.alpha.2 peptide. In some embodiments,
the adjuvant generates an immune response to the EphA2 and/or
IL-13R.alpha.2 peptide and does not produce an allergy or other
adverse reaction. Adjuvants can enhance an immune response by
several mechanisms including, e.g., lymphocyte recruitment,
stimulation of B and/or T cells, stimulation of dendritic cells and
stimulation of macrophages.
[0100] Specific examples of adjuvants include, but are not limited
to, Montanide ISA-51, Montanide ISA 50V, Montanide, ISA 206,
Montanide IMS 1312, Vaxlmmune.RTM. (CpG7909; Coley
Pharmaceuticals), aluminum salts (alum) (such as aluminum
hydroxide, aluminum phosphate, and aluminum sulfate), 3
De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MFS9
(Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline),
polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine
compounds (see International Application No. PCT/US2007/064857,
published as International Publication No. WO2007/109812),
imidazoquinoxaline compounds (see International Application No.
PCT/US2007/064858, published as International Publication No.
WO2007/109813) and saponins, such as QS21 (see Kensil et al., in
Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell
& Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). In
some embodiments, the adjuvant is Freund's adjuvant (complete or
incomplete). Other adjuvants are oil in water emulsions (such as
squalene or peanut oil), optionally in combination with immune
stimulants, such as monophosphoryl lipid A (see Stoute et al., N.
Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld
Today, Nov. 15, 1998). Such adjuvants can be used with or without
other specific immunostimulating agents such as MPL or 3-DMP, QS21,
polymeric or monomeric amino acids such as polyglutamic acid or
polylysine, or other immunopotentiating agents. It should be
understood that different formulations of EphA2 peptides may
comprise different adjuvants or may comprise the same adjuvant.
5.6 Helper T Cell Epitopes
[0101] In some embodiments, the EphA2 and/or IL-13R.alpha.2
peptides provided herein are administered concurrently with a
helper T cell epitope. Helper T cell epitopes include agents that
are capable of inducing a helper T cell response by the immune
system. Helper T cells are CD4+ T cells. In some embodiments,
helper T cell epitopes are presented by Class II MHC molecules, and
may be recognized by the T cell receptor (TCR) of helper T cells
(CD4+ T cells), thereby activating the CD4+ T cells, causing them
to proliferate, secrete cytokines such as IL2, and activate
professional antigen presenting cells. Through a variety of
mechanisms, activated helper T cells also stimulate killer T cells
(also known as CD8+ T cells), thereby prolonging and increasing the
CD8+ T cell response. Exemplary helper T cell epitopes that can be
administered concurrently with the EphA2 peptides provided herein
include, without limitation, PADRE (see, e.g., Alexander et al,
Immunity, 1:751-761, 1994), HBVcore.sub.128-140, and tetanus
toxoid.
5.7 Production and Purification of epha2 Peptides
[0102] The EphA2 and/or IL-13R.alpha.2 peptides described herein
can be made by standard recombinant DNA techniques or by protein
synthetic techniques, e.g., by use of a peptide synthesizer. For
example, a nucleic acid molecule encoding an EphA2 and/or
IL-13R.alpha.2 peptide can be synthesized by conventional
techniques including automated DNA synthesizers. As another
example, the EphA2 and/or IL-13R.alpha.2 peptides described herein
may be generated using conventional step-wise solution or solid
phase synthesis (see, e.g., Chemical Approaches to the Synthesis of
Peptides and Proteins, Williams et al., Eds., 1997, CRC Press, Boca
Raton Fla., and references cited therein; Solid Phase Peptide
Synthesis: A Practical Approach, Atherton & Sheppard, Eds.,
1989, IRL Press, Oxford, England, and references cited therein) or
through the use of segment condensation (see, e.g., Liu et al.,
1996, Tetrahedron Lett. 37(7):933-936; Baca, et al., 1995, J. Am.
Chem. Soc. 117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein
Res. 45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu
and Tam, 1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam,
1994, Proc. Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li,
1988, Int. J. Peptide Protein Res. 31:322-334).
[0103] The EphA2 or IL-13R.alpha.2 peptides described herein may be
obtained from any information available to those of skill in the
art (i.e., from Genbank, the literature, or by routine cloning). A
nucleotide sequence coding for an EphA2 or IL-13R.alpha.2 peptide
can be inserted into an appropriate expression vector, i.e., a
vector which contains the necessary elements for the transcription
and translation of the inserted protein-coding sequence. A variety
of host-vector systems may be utilized to express the
protein-coding sequence. These include but are not limited to
mammalian cell systems infected with virus (e.g., vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.,
baculovirus); microorganisms such as yeast (e.g. Pichia) containing
yeast vectors; or bacteria (such as E. coli) transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression
elements of vectors vary in their strengths and specificities.
Depending on the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be used. In
a specific embodiment, the peptide is expressed in E. coli. In
another specific embodiment, the peptide is expressed in
Pichia.
[0104] Once an EphA2 or IL-13R.alpha.2 peptide has been produced by
recombinant expression or by chemical synthesis, it may be purified
by any method known in the art for purification of a protein, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
5.8 Pharmaceutical Compositions and Routes of Administration
[0105] Provided herein are pharmaceutical compositions comprising
EphA2 and/or IL-13R.alpha.2 eptides for use in the methods
described herein. In certain embodiments, a composition provided
herein comprises EphA2 and/or IL-13R.alpha.2 peptide and one or
more additional peptides or agents. In certain embodiments, the
compositions provided herein comprise an EphA2 and/or
IL-13R.alpha.2 peptide and a helper T cell epitope, an adjuvant,
and/or an immune response modifier. The pharmaceutical compositions
provided herein are suitable for veterinary and/or human
administration.
[0106] The pharmaceutical compositions provided herein can be in
any form that allows for the composition to be administered to a
subject, said subject preferably being an animal, including, but
not limited to a human, mammal, or non-human animal, such as a cow,
horse, sheep, pig, fowl, cat, dog, mouse, rat, rabbit, guinea pig,
etc., and is more preferably a mammal, and most preferably a
human.
[0107] In specific embodiments, the compositions provided herein
are in the form of a liquid (e.g., an elixir, syrup, solution,
emulsion, or suspension). Typical routes of administration of the
liquid compositions provided herein may include, without
limitation, parenteral, intradermal, intratumoral, intracerebral,
and intrathecal. Parenteral administration includes, without
limitation, subcutaneous, intranodal, intravenous, intramuscular,
intraperitoneal, and intrapleural administration techniques. In a
specific embodiment, the compositions are administered
parenterally. In a composition for administration by injection, one
or more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer, and isotonic agent may
be included. In a specific embodiment, a pump may be used to
deliver the vaccines (see, e.g, Sefton, CRC Crit. Ref. Biomed. Eng.
1987, 14, 201; Buchwald et al., Surgery 1980, 88: 507; Saudek et
al., N. Engl. J. Med. 1989, 321: 574). In a specific embodiment,
the pump may be, but is not limited to, an insulin-like pump.
[0108] Materials used in preparing the pharmaceutical compositions
provided herein can be non-toxic in the amounts used. It may be
evident to those of ordinary skill in the art that the optimal
dosage of the active ingredient(s) in the pharmaceutical
composition will depend on a variety of factors. Relevant factors
include, without limitation, the type of subject (e.g., human), the
overall health of the subject, the type of cancer the subject is in
need of treatment of, the use of the composition as part of a
multi-drug regimen, the particular form of the peptide being
administered, the manner of administration, and the composition
employed.
[0109] The liquid compositions provided herein, whether they are
solutions, suspensions, or other like form, can also include one or
more of the following: sterile diluents such as water for
injection, saline solution, preferably physiological saline,
Ringer's solution, isotonic sodium chloride, fixed oils such as
synthetic mono or digylcerides which can serve as the solvent or
suspending medium, polyethylene glycols, glycerin, cyclodextrin,
propylene glycol, or other solvents; antibacterial agents such as
benzyl alcohol or methyl paraben; antioxidants such as ascorbic
acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates,
citrates, or phosphates; and agents for the adjustment of tonicity
such as sodium chloride or dextrose. A parenteral composition can
be enclosed in an ampoule, a disposable syringe, or a multiple-dose
vial made of glass, plastic or other material. An injectable
composition is preferably sterile.
[0110] The compositions provided herein may comprise a
pharmaceutically acceptable carrier or vehicle. As used herein, the
term "pharmaceutically acceptable" means approved by a regulatory
agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeiae for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant, excipient, or vehicle with which the
pharmaceutical composition is administered. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. Examples of
suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E.W. Martin. The formulation should
suit the mode of administration.
[0111] In one embodiment, the compositions provided herein are
formulated in accordance with routine procedures as a
pharmaceutical composition adapted for parenteral administration to
animals, particularly human beings. Generally, the ingredients in
the compositions are supplied either separately or mixed together
in unit dosage form, for example, as a dry lyophilized powder or
water free concentrate in a hermetically sealed container such as
an ampoule or sachet indicating the quantity of active agent. Where
a composition described herein is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients can be mixed prior to administration, if
necessary.
[0112] The compositions described herein can comprise an additional
active agent selected from among those including, but not limited
to, an additional prophylactic agent, an additional therapeutic
agent, an antiemetic agent, a hematopoietic colony stimulating
factor, an adjuvant therapy, an antibody/antibody fragment-based
agent, an anti-depressant and an analgesic agent. In specific
embodiments, the additional active agent is a second EphA2 and/or
IL-13R.alpha.2 peptide (i.e., an EphA2 and/or IL-13R.alpha.2
peptide different from the one that forms the base of the
composition). In specific embodiments, the additional active agent
is a second peptide that is not an EphA2 and/or IL-13R.alpha.2
peptide.
[0113] The pharmaceutical compositions provided herein can be
prepared using methodology well known in the pharmaceutical art.
For example, a composition intended to be administered by injection
can be prepared by combining the EphA2 and/or IL-13R.alpha.2
peptides described herein with water and/or other liquid components
so as to form a solution. A surfactant can be added to facilitate
the formation of a homogeneous solution or suspension.
[0114] The pharmaceutical compositions described herein can be
included in a container, pack, or dispenser together with
instructions for administration.
5.8.1 Dosage and Frequency of Administration
[0115] The amount of a composition described herein (e.g., a
composition comprising an EphA2 and/or IL-13R.alpha.2 peptide; a
composition comprising an EphA2 peptide and an IL-13R.alpha.2
peptide a composition comprising an EphA2 and/or IL-13R.alpha.2
peptide and a helper T cell epitope, an adjuvant) which will be
effective in the treatment, prevention, and or management of cancer
may depend on the status of the cancer, the patient to whom the
composition(s) is to be administered, the route of administration,
and/or the type of cancer. Such doses can be determined by standard
clinical techniques and may be decided according to the judgment of
the practitioner.
[0116] For example, effective doses may vary depending upon means
of administration, target site, physiological state of the patient
(including age, body weight, health), whether the patient is human
or an animal, other medications administered, and whether treatment
is prophylactic or therapeutic. Usually, the patient is a human but
nonhuman mammals including transgenic mammals can also be treated.
Treatment dosages are optimally titrated to optimize safety and
efficacy.
[0117] In certain embodiments, an in vitro assay is employed to
help identify optimal dosage ranges. Effective doses may be
extrapolated from dose response curves derived from in vitro or
animal model test systems.
[0118] In certain embodiments, a composition comprises about 25,
50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, 500, 550, 600, 650, 700, 750, or 800 .mu.g of
an EphA2 peptide per dose. In other embodiments, compositions
comprise about 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 to 150,
50 to 200, 100 to 150, 100 to 200, 100 to 250, 100 to 300, 150 to
200, 150 to 250, 150 to 300, 200 to 250, 250 to 300, 250 to 350,
250 to 400, 300 to 350, 300 to 400, 300 to 450, 300 to 500, 350 to
400, 350 to 450, 400 to 500, 400 to 600, 500 to 600, 500 to 700,
600 to 700, 600 to 800, or 700 to 800 .mu.g of an EphA2 and/or
IL-13R.alpha.2 peptide per dose. In other embodiments, compositions
comprise about 5 .mu.g to 100 mg, 15 .mu.g to 50 mg, 15 .mu.g to 25
mg, 15 .mu.g to 10 mg, 15 .mu.g to 5 mg, 15 .mu.g to 1 mg, 15 .mu.g
to 100 .mu.g, 15 .mu.g to 75 .mu.g, 5 .mu.g to 50 .mu.g, 10 .mu.g
to 50 .mu.g, 15 .mu.g to 45 .mu.g, 20 .mu.g to 40 .mu.g, or 25 to
35 .mu.g of an EphA2 and/or IL-13R.alpha.2 peptide per kilogram of
the patient.
[0119] In certain embodiments, compositions comprising an EphA2
and/or IL-13R.alpha.2 peptide are administered concurrently with a
helper T cell epitope. In some embodiments, such compositions are
administered concurrently with about 25, 50, 75, 100, 125, 150,
175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,
500, 550, or 600 .mu.g of a helper T cell epitope. In other
embodiments, such compositions are administered concurrently with
about 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 to 150, 50 to
200, 100 to 150, 100 to 200, 100 to 250, 100 to 300, 150 to 200,
150 to 250, 150 to 300, 200 to 250, 250 to 300, 250 to 350, 250 to
400, 300 to 350, 300 to 400, 300 to 450, 300 to 500, 350 to 400,
350 to 450, 400 to 500, 400 to 600, or 500 to 600 .mu.g of a helper
T cell epitope.
[0120] In certain embodiments, the compositions comprising an EphA2
and/or IL-13R.alpha.2 peptide are administered concurrently with an
immune response modifier, e.g., about 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or
1800 .mu.g of an immune response modifier; or about 100 to 300, 200
to 400, 400 to 800, 600 to 800, 800 to 1000, 800 to 1200, 1000 to
1200, 1000 to 1400, 1200 to 1400, 1200 to 1600, 1400 to 1600, 1400
to 1800, or 1600 to 1800 .mu.g of an immune response modifier.
[0121] In certain embodiments, the compositions comprising an EphA2
and/or IL-13R.alpha.2 peptide are administered concurrently with an
adjuvant. In some embodiments, the compositions comprising an EphA2
peptide are mixed 0.5 to 1, 1 to 0.5, 1 to 1, 1 to 2, 1 to 3, 2 to
1, or 3 to 1 with an adjuvant.
[0122] In certain embodiments, a composition described herein is
administered to a subject once as a single dose. In some
embodiments, a composition described herein is administered in
multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than
10 doses), wherein the doses may be separated by at least 1 day, 2
days, 3 days, 4, days 5 days, 6 days, 7 days, 8 days, 9 days, 10
days, 15 days, or 30 days.
[0123] In some embodiments, a composition described herein is
administered over the course of 21 weeks, with administrations
occurring on weeks 0, 3, 6, 9, 12, 15, 18 and 21. In certain
embodiments, the composition is administered concurrently with a
helper T cell epitope, an adjuvant, and/or an immune response
modifer. In a specific embodiment, a composition described herein
comprising is administered over the course of 21 weeks, with
administrations occurring on weeks 0, 3, 6, 9, 12, 15, 18 and 21,
and the composition is administered concurrently with an immune
response modifier, wherein the immune response modifier is
administered on the day of each administration of the composition
comprising an EphA2 and/or IL-13R.alpha.2 peptide and on day 4
after each administration of the composition comprising an EphA2
and/or IL-13R.alpha.2 peptide. In another specific embodiment, a
composition described herein is administered over the course of 21
weeks, with administrations occurring on weeks 0, 3, 6, 9, 12, 15,
18 and 21, and the composition is administered concurrently with an
immune response modifier, wherein the immune response modifier is
administered on the day of each administration of the composition
comprising an EphA2 and/or IL-13R.alpha.2 peptide.
5.8.2 Patient Populations
[0124] In certain an EphA2 and/or IL-13R.alpha.2 peptide or
composition thereof may be administered to a naive subject, i.e., a
subject that does not have cancer. In one embodiment, an EphA2
and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a naive subject that is at risk of acquiring
cancer.
[0125] In certain embodiments, an EphA2 and/or IL-13R.alpha.2
peptide or composition thereof is administered to a patient who has
been diagnosed with cancer. In some embodiments, an EphA2 and/or
IL-13R.alpha.2 peptide or composition thereof is administered to a
patient with cancer before symptoms manifest or symptoms become
severe. In a specific embodiment, the cancer is brain cancer.
[0126] In certain embodiments, an EphA2 and/or IL-13R.alpha.2
peptide or composition thereof is administered to a patient who is
in need of treatment, prevention, and/or management of cancer. Such
subjects may or may not have been previously treated for cancer or
may be in remission, relapsed, or may have failed treatment. Such
patients may also have abnormal cytogenetics.
[0127] In a specific embodiment, the subject has been diagnosed
with cancer using techniques known to one of skill in the art
including, but not limited to, neurological examination; imaging
methods (e.g., computed tomography (CT), magnetic resonance imaging
(MRI), ultrasound, X-ray imaging, fluid-attenuated
inversion-recovery (FLAIR) sequences, T2 weighted imaging, and
positron emission tomography (PET) scans); and biopsy (e.g.,
sterotactic biopsy). Tumor response to therapy may be evaluated by
McDonald criteria or Response assesment in neuro-oncology (RANO)
criteria. Tumor size or response to treatment can be evaluated by
various magnetic resonance imaging techniques including
diffusion-weighted imaging, perfusion-weighted imaging, dynamic
contrast-enhanced T1 permeability imaging, dynamic susceptibility
contrast, diffusion-tensor imaging, and magnetic resonance
spectroscopy, anatomic MRI T2-weighted images, fluid attenuated
inversion recovery (FLAIR) T2-weighted images, and
gadolinium-enhanced T1-weighted images. These imagining techniques
can be used to assess tumor cellularity, white matter invasion,
metabolic derangement including hypoxia and necrosis, neovascular
capillary blood volume, or permeability. Positron emission
tomograph (PET) technology can also be used to image tumor
response, such as 18F-fluoromisonidazole PET and
3'-deoxy-3'-18F-fluorothymidine PET.
[0128] In some embodiments, an EphA2 and/or IL-13R.alpha.2 peptide
or composition thereof is administered to a subject that is in
remission from brain cancer. In a specific embodiment, the subject
has no detectable brain cancer, i.e., no brain cancer is detectable
using a conventional method described herein (e.g., MRI) or known
to one of skill in the art.
[0129] In one embodiment, an EphA2 and/or IL-13R.alpha.2 peptide or
composition thereof is administered to a subject diagnosed with
glioma. In a specific embodiment, an EphA2 and/or IL-13R.alpha.2
peptide or composition thereof is administered to a subject
diagnosed with astrocytoma (e.g., pilocytic astrocytoma, diffuse
astrocytoma, and anaplastic astrocytoma). In another specific
embodiment, an EphA2 and/or IL-13R.alpha.2 peptide or composition
thereof is administered to a subject diagnosed with glioblastoma.
In another specific embodiment, an EphA2 and/or IL-13R.alpha.2
peptide or composition thereof is administered to a subject
diagnosed with oligodendroglioma. In another specific embodiment,
an EphA2 and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with brain stem glioma. In
another specific embodiment, vis administered to a subject
diagnosed with ependymoma. In another specific embodiment, an EphA2
and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with a mixed tumor comprising
more than one glial cell types.
[0130] In a specific embodiment, an EphA2 and/or IL-13R.alpha.2
peptide or composition thereof is administered to a subject
diagnosed with recurrent malignant glioma. In another specific
embodiment, an EphA2 and/or IL-13R.alpha.2 peptide or composition
thereof is administered to a subject diagnosed with high-risk WHO
Grade II Astrocytomas. In another specific embodiment, an EphA2
and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with Oligo Astrocytoma. In
another specific embodiment, an EphA2 and/or IL-13R.alpha.2 peptide
or composition thereof is administered to a subject diagnosed with
recurrent WHO Grade II Glioma. In another specific embodiment, an
EphA2 and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with newly-diagnosed malignant
or intrinsic brain stem glioma. In another specific embodiment, an
EphA2 and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with incompletely resected
non-brainstem glioma. In another specific embodiment, an EphA2
and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with recurrent unresectable
low-grade glioma.
[0131] In a specific embodiment, an EphA2 and/or IL-13R.alpha.2
peptide or composition thereof is administered to a subject
diagnosed with acoustic schwannoma. In another specific embodiment,
an EphA2 and/or IL-13R.alpha.2 peptide or composition thereof is
administered to a subject diagnosed with cranial pharyngioma. In
another specific embodiment, an EphA2 and/or IL-13R.alpha.2 peptide
or composition thereof is administered to a subject diagnosed with
meningioma. In another specific embodiment, an EphA2 and/or
IL-13R.alpha.2 peptide or composition thereof is administered to a
subject diagnosed with medulloblastoma. In another specific
embodiment, an EphA2 and/or IL-13R.alpha.2 peptide or composition
thereof described herein is administered to a subject diagnosed
with primary central nervous system lymphoma. In another specific
embodiment, an EphA2 and/or IL-13R.alpha.2 peptide or composition
thereof is administered to a subject diagnosed with a tumor of the
pineal gland (e.g., a pineal astrocytic tumor or a pineal
parenchymal tumor). In another specific embodiment, an EphA2 and/or
IL-13R.alpha.2 peptide or composition thereof is administered to a
subject diagnosed with a tumor of the pituitary gland.
5.8.3 Combination Therapies
[0132] In certain embodiments, the methods provided herein for
preventing, treating, and/or managing cancer comprise administering
to a patient (e.g., a human patient) in need thereof a
prophylactically and/or a therapeutically effective regimen, the
regimen comprising administering to the patient EphA2 and/or
IL-13R.alpha.2 peptide or composition thereof described herein and
one or more additional therapies. An EphA2 peptide or composition
thereof described herein and an additional therapy can be
administered separately, concurrently, or sequentially. The
combination therapies can act additively or synergistically. In a
specific embodiment, a combination therapy provided herein
comprises an EphA2 peptide and an IL-13R.alpha.2.
[0133] The combination therapies can be administered to a subject
in the same pharmaceutical composition. Alternatively, the
combination therapies can be administered concurrently to a subject
in separate pharmaceutical compositions. The combination therapies
may be administered to a subject by the same or different routes of
administration.
[0134] Any therapy (e.g., therapeutic or prophylactic agent) which
is useful, has been used, or is currently being used for the
prevention, treatment, and/or management of cancer brain cancer)
can be used in combination with an EphA2 and/or IL-13R.alpha.2
peptide or composition described herein in the methods described
herein. Therapies include, but are not limited to, peptides,
polypeptides, antibodies, conjugates, nucleic acid molecules, small
molecules, mimetic agents, synthetic drugs, inorganic molecules,
and organic molecules. Non-limiting examples of cancer therapies
include chemotherapy, radiation therapy, hormonal therapy, surgery,
small molecule therapy, anti-angiogenic therapy, differentiation
therapy, epigenetic therapy, radioimmunotherapy, targeted therapy,
and/or biological therapy including immunotherapy. In certain
embodiments, a prophylactically and/or therapeutically effective
regimen of the invention comprises the administration of a
combination of therapies.
[0135] Examples of cancer therapies which can be used in
combination with EphA2 and/or IL-13R.alpha.2 peptide or composition
thereof described herein in accordance with the methods described
herein include, but are not limited to: acivicin; aclarubicin;
acodazole hydrochloride; acronine; adozelesin; aldesleukin;
altretamine; ambomycin; ametantrone acetate; aminoglutethimide;
amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;
asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;
benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide
dimesylate; bisphosphonates (e.g., pamidronate (Aredria), sodium
clondronate (Bonefos), zoledronic acid (Zometa), alendronate
(Fosamax), etidronate, ibandornate, cimadronate, risedromate, and
tiludromate); bizelesin; bleomycin sulfate; brequinar sodium;
bropirimine; busulfan; cactinomycin; calusterone; caracemide;
carbetimer; carboplatin; carmustine; carubicin hydrochloride;
carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin;
cladribine; crisnatol mesylate; cyclophosphamide; cytarabine
(Ara-C); dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine (Dacogen); demethylation agents, dexormaplatin;
dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;
doxonibicin; doxorubicin hydrochloride; droloxifene; droloxifene
citrate; dromostanolone propionate; duazomycin; edatrexate;
eflornithine hydrochloride; EphA2 inhibitors; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; histone deacetylase
inhibitors (HDACs) gemcitabine hydrochloride; hydroxyurea;
idarubicin hydrochloride; ifosfamide; ilmofosine; imatinib mesylate
(Gleevec, Glivec); interleukin II (including recombinant
interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b;
interferon alpha-n1; interferon alpha-n3; interferon beta-I a;
interferon gamma-I b; iproplatin; irinotecan hydrochloride;
lanreotide acetate; lenalidomide (Revlimid); letrozole; leuprolide
acetate; liarozole hydrochloride; lometrexol sodium; lomustine;
losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; anti-CD2 antibodies (e.g., siplizumab (MedImmune
Inc.; International Publication No. WO 02/098370, which is
incorporated herein by reference in its entirety)); megestrol
acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxaliplatin; oxisuran; paclitaxel; pegaspargase;
peliomycin; pentamustine; peplomycin sulfate; perfosfamide;
pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin;
plomestane; porfimer sodium; porfiromycin; prednimustine;
procarbazine hydrochloride; puromycin; puromycin hydrochloride;
pyrazofiirin; riboprine; rogletimide; safingol; safingol
hydrochloride; semustine; simtrazene; sparfosate sodium;
sparsomycin; spirogermanium hydrochloride; spiromustine;
spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;
tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate
glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine
sulfate; vindesine; vindesine sulfate; vinepidine sulfate;
vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride.
[0136] Other examples of cancer therapies which can be used in
combination with an EphA2 and/or IL-13R.alpha.2 peptide or
composition thereof described herein in accordance with the methods
described herein include, but are not limited to: 20-epi-1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;
acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK
antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate: exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; aemcitabine;
glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,
atorvastatin, cerivastatin, fluvastatin, lescol, lupitor,
lovastatin, rosuvastatin, and simvastatin); hepsulfam; heregulin;
hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin;
idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;
imiquimod; immunostimulant peptides; insulin-like growth factor-1
receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole; LFA-3TIP
(Biogen, Cambridge, Mass.; International Publication No. WO 93/0686
and U.S. Pat. No. 6,162,432); liarozole; linear polyamine analogue;
lipophilic disaccharide peptide; lipophilic platinum compounds;
lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium
texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;
marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors; menogaril; merbarone; meterelin;
methioninase; metoclopramide; MIF inhibitor; mifepristone;
miltefosine; mirimostim; mismatched double stranded RNA;
mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin
fibroblast growth factor-saporin; mitoxantrone; mofarotene;
molgramostim; monoclonal antibody, human chorionic gonadotrophin;
monophosphoryl lipid A+myobacterium cell wall sk; mopidamol;
multiple drug resistance gene inhibitor; multiple tumor suppressor
1-based therapy; mustard anticancer agent; mycaperoxide B;
mycobacterial cell wall extract; myriaporone; N-acetyldinaline;
N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;
nilutamide; nisamycin; nitric oxide modulators; nitroxide
antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin;
oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RH retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;
tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase
inhibitors; temoporfin; temozolomide; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin
receptor agonist; thymotrinan; thyroid stimulating hormone; tin
ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; totipotent stem cell factor; translation inhibitors;
tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived
growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin B; vector system, erythrocyte gene therapy;
thalidomide; velaresol; veramine; verdins; verteporfin;
vinorelbine; vinxaltine; VITAXIN.TM. (see U.S. Patent Pub. No. US
2002/0168360 A1 dated Nov. 14, 2002, entitled "Methods of
Preventing or Treating Inflammatory or Autoimmune Disorders by
Administering Integrin av.beta.3 Antagonists in Combination With
Other Prophylactic or Therapeutic Agents"): vorozole; zanoterone;
zeniplatin; zilascorb; and zinostatin stimalamer.
[0137] In some embodiments, the therapy(ies) used in combination
with an EphA2 and/or IL-13R.alpha.2 peptide or composition thereof
described herein in accordance with the methods described herein is
an immunomodulatory agent. Non-limiting examples of
immunomodulatory agents include proteinaceous agents such as
cytokines, peptide mimetics, and antibodies (e.g., human,
humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or
F(ab)2 fragments or epitope binding fragments), nucleic acid
molecules (e.g., antisense nucleic acid molecules and triple
helices), small molecules, organic compounds, and inorganic
compounds. In particular, immunomodulatory agents include, but are
not limited to, methotrexate, leflunomide, cyclophosphamide,
cytoxan, Immuran, cyclosporine A, minocycline, azathioprine,
antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP),
corticosteroids, steroids, mycophenolate mofetil, rapamycin
(sirolimus), mizoribine, deoxyspergualin, brequinar,
malononitriloamindes (e.g., leflunomide), T cell receptor
modulators, cytokine receptor modulators, and modulators mast cell
modulators. Other examples of immunomodulatory agents can be found,
e.g., in U.S. Publication No. 2005/0002934 A1 at paragraphs 259-275
which is incorporated herein by reference in its entirety. In one
embodiment, the immunomodulatory agent is a chemotherapeutic agent.
In an alternative embodiment, the immunomodulatory agent is an
immunomodulatory agent other than a chemotherapeutic agent. In some
embodiments, the therapy(ies) used in accordance with the invention
is not an immunomodulatory agent.
[0138] In some embodiments, the therapy(ies) used in combination
with an EphA2 and/or IL-13R.alpha.2 peptide or composition thereof
described herein in accordance with the methods described herein is
an anti-angiogenic agent. Non-limiting examples of anti-angiogenic
agents include proteins, polypeptides, peptides, conjugates,
antibodies (e.g., human, humanized, chimeric, monoclonal,
polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2 fragments, and
antigen-binding fragments thereof) such as antibodies that
specifically bind to TNF-.alpha., nucleic acid molecules (e.g.,
antisense molecules or triple helices), organic molecules,
inorganic molecules, and small molecules that reduce or inhibit
angiogenesis. Other examples of anti-angiogenic agents can be
found, e.g., in U.S. Publication No. 2005/0002934 A1 at paragraphs
277-282, which is incorporated by reference in its entirety. In a
preferred embodiment, the anti-angiogenic therapy is bevacizumab
(Avastin.RTM.). In other embodiments, the therapy(ies) used in
accordance with the invention is not an anti-angiogenic agent.
[0139] In some embodiments, the therapy(ies) used in combination
with an EphA2 and/or IL-13R.alpha.2 peptide or composition thereof
described herein in accordance with the methods described herein is
an anti-inflammatory agent. Non-limiting examples of
anti-inflammatory agents include any anti-inflammatory agent,
including agents useful in therapies for inflammatory disorders,
well-known to one of skill in the art. Non-limiting examples of
anti-inflammatory agents include non-steroidal anti-inflammatory
drugs (NSAIDs), steroidal anti-inflammatory drugs, anticholinergics
(e.g., atropine sulfate, atropine methylnitrate, and ipratropium
bromide (ATROVENT.TM.)), beta2-agonists (e.g., abuterol
(VENTOLIN.TM. and PROVENTIL.TM.), bitolterol (TORNALATE.TM.),
levalbuterol (XOPONEX.TM.), metaproterenol (ALUPENT.TM.),
pirbuterol (MAXAIR.TM.), terbutlaine (BRETHAIRE.TM. and
BRETHINE.TM.), albuterol (PROVENTIL.TM., REPETABS.TM., and
VOLMAX.TM.), formoterol (FORADIL AEROLIZER.TM.), and salmeterol
(SEREVENT.TM. and SEREVENT DISKUS.TM.)), and methylxanthines (e.g.,
theophylline (UNIPHYL.TM., THEO-DUR.TM., SLO-BID.TM., AND
TEHO-42.TM.)). Examples of NSAIDs include, but are not limited to,
aspirin, ibuprofen, celecoxib (CELEBREX.TM.), diclofenac
(VOLTAREN.TM.), etodolac (LODINE.TM.), fenoprofen (NALFON.TM.),
indomethacin (INDOCIN.TM.), ketoralac (TORADOL.TM.), oxaprozin
(DAYPRO.TM.), nabumentone (RELAFEN.TM.), sulindac (CLINORIL.TM.),
tolmentin (TOLECTIN.TM.), rofecoxib (VIOXX.TM.), naproxen
(ALEVE.TM., NAPROSYN.TM.), ketoprofen (ACTRON.TM.) and nabumetone
(RELAFEN.TM.). Such NSAIDs function by inhibiting a cyclooxgenase
enzyme (e.g., COX-1 and/or COX-2). Examples of steroidal
anti-inflammatory drugs include, but are not limited to,
glucocorticoids, dexamethasone (DECADRON.TM.), corticosteroids
(e.g., methylprednisolone (MEDROL.TM.)), cortisone, hydrocortisone,
prednisone (PREDNISONE.TM. and DELTASONE.TM.), prednisolone
(PRELONE.TM. and PEDIAPRED.TM.) triamcinolone, azulfidine, and
inhibitors of eicosanoids (e.g., prostaglandins, thromboxanes, and
leukotrienes. Other examples of anti-inflammatory agents can be
found, e.g., in U.S. Publication No. 005/0002934 A1 at paragraphs
290-294, which is incorporated by reference in its entirety. In
other embodiments, the therapy(ies) used in accordance with the
invention is not an anti-inflammatory agent.
[0140] In certain embodiments, the therapy(ies) used in combination
with an EphA2 and/or IL-13R.alpha.2 peptide or composition thereof
described herein in accordance with the methods described herein is
an alkylating agent, a nitrosourea, an antimetabolite, and
anthracyclin, a topoisomerase II inhibitor, or a mitotic inhibitor.
Alkylating agents include, but are not limited to, busulfan,
cisplatin, carboplatin, cholormbucil, cyclophosphamide, ifosfamide,
decarbazine, mechlorethamine, melphalan, and temozolomide.
Nitrosoureas include, but are not limited to carmustine (BCNU) and
lomustine (CCNU). Antimetabolites include but are not limited to
5-fluorouracil, capecitabine, methotrexate, gemcitabine,
cytarabine, and fludarabine. Anthracyclins include but are not
limited to daunorubicin, doxorubicin, epirubicin, idarubicin, and
mitoxantrone. Topoisomerase II inhibitors include, but are not
limited to, topotecan, irinotecan, etopiside (VP-16), and
teniposide. Mitotic inhibitors include, but are not limited to
taxanes (paclitaxel, docetaxel), and the vinca alkaloids
(vinblastine, vincristine, and vinorelbine).
[0141] Currently available cancer therapies and their dosages,
routes of administration and recommended usage are known in the art
and have been described in such literature as the Physician's Desk
Reference (60th ed., 2006).
6. EXAMPLES
[0142] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
6.1 Example 1
[0143] This example demonstrates that EphA2 and IL-13R.alpha.2 are
cancer stem cell antigens.
6.1.1 Materials and Methods
[0144] Flow cytometry was performed on the brain cancer cell line
A-172 to assess the expression of EphA2 and IL-13R.alpha.2 on these
cancer cells. The experimental protocol included the following
steps.
[0145] A-172 cells were thawed and plated in 10 cm culture dishes
under sterile conditions and using aseptic technique. The A-172
cells were grown in MEM containing 10% FBS. Both cell lines were
grown at 37.degree. C. with 5% CO.sub.2 in humidified air. The
A-172 cells were passaged 1:5 every 3 days.
[0146] On the day of the experiments, the cells were washed once
with 1.times.PBS and incubated for 3 minutes with 2 ml 0.25%
trypsin-EDTA at 37.degree. C. The cells were then detached from the
tissue culture plates with gentle agitation and diluted with 10 ml
of DMEM. The cells then were placed in a 50 ml conical tube and
centrifuged at 350.times.g for 5 minutes. The supernatant was
aspirated and the cells were resuspended in 10 ml DMEM. Fifty .mu.l
of the cells were mixed with an equal volume of trypan blue and the
mixture was carefully placed on a hemacytometer for counting. The
cell volumes were then adjusted with DMEM to a concentration of
5.times.10.sup.6/ml.
[0147] Twenty flow cytometry tubes Fisher Scientific) were prepared
and 100 .mu.l of the cells were added to each tube
(5.times.10.sup.5 cells/tube) (10 tubes with A-172 cells).
[0148] Twenty .mu.l of Fc blocking reagent was added to each tube
and the tubes were incubated at room temperature for 10
minutes.
[0149] Ten .mu.l of each antibody, as provided in Table 1, below,
was diluted to the described working concentration provided in
Table 2, below, and was added to each appropriate tube. The tubes
were incubated for 30 minutes at 4.degree. C. with gentle
agitation.
TABLE-US-00001 TABLE 1 A-172 CELLS Tube #1 #2-3 #4-5 #6 #7 #8 #9
#10 Primary Unstained Isotype Secondary .alpha.-CD133
.alpha.-IL13R.alpha.2 .alpha.-EphA2 .alpha.-CD133 + .alpha.-CD133 +
Antibody control Antibodies .alpha.-IL13R.alpha.2 .alpha.-EphA2
Alone Secondary Anti-mouse Anti-mouse Anti-mouse Anti-goat
Anti-goat Anti-mouse + Anti-mouse + Antibody OR OR Anti-goat
Anti-goat Anti-goat Anti-goat
TABLE-US-00002 TABLE 2 Antibody Working Concentration CD133 16.5
.mu.g/ml IL13R.alpha.2 10 .mu.g/ml EphA2 50 .mu.g/ml Anti-mouse-APC
1:200 Anti-goat-FITC 1:200
[0150] After the incubation, the cells were centrifuged at
300.times.g for 1 minute in a tabletop, refrigerated
microcentrifuge. The supernatant was removed and the cells were
washed with ice cold FACS buffer 3 times. The cells were then
resuspended in 100 .mu.l of FACS buffer and 10 .mu.l of the
secondary antibodies was added to the appropriate tubes. The tubes
were incubated for 30 minutes at 4.degree. C. with gentle agitation
in the dark.
[0151] After the incubation, the cells were centrifuged at
300.times.g for 1 minute in a tabletop, refrigerated
microcentrifuge. The supernatant was removed and the cells were
washed with ice cold FACS buffer 3 times. The cells were then
resuspended in 200 .mu.l of FACS buffer and analyzed on a
FACSCalibur (BD Biosciences) flow cytometer.
6.1.2 Results
[0152] In brain cancer, the brain cancer stem cells can be
identified using the marker CD133, i.e., brain cancer stem cells
are known to express the CD133 antigen (see, e.g., Singh et al.,
2004, Nature 432:396-401, the disclosure of which is hereby
incorporated by reference in its entirety). The cancer stem cells
of the brain cancer cell line A-172 express CD133 (see, e.g., Qiang
et al., 2009, Cancer Letters 271:13-21, the disclosures of which is
hereby incorporated by reference in its entirety).
[0153] As demonstrated in FIG. 1, all cells of the A-172 line were
positive for EphA2 and IL-13R.alpha.2, whereas a small population
of such cells also were positive for CD133. This CD133+ cell
subpopulation thus represents the cancer stem cell subpopulation of
the A-172 cell line, and the same expression pattern of CD133 on
A-172 cells was observed in a subsequent duplicate experiment (see
FIGS. 6 and 7).
[0154] As demonstrated in FIG. 2, the CD 133+ population also was
positive for expression of EphA2, thus demonstrating that EphA2 is
present on the cancer stem cell population obtained from the A-172
cell line, and thus that EphA2 is a cancer stem cell antigen. This
fact was verified in a subsequent duplicate experiment (see FIG.
8). Moreover, as shown in FIG. 4, EphA2 was expressed to higher
levels on on CD133+Cells as compared to CD133- cells
[0155] Similarly, as demonstrated by FIG. 2, the CD133+ population
of A-172 cell line also was positive for expression of
IL-13R.alpha.2, thus demonstrating that IL-13R.alpha.2 is present
on the cancer stem cell population obtained from the A-172 cell
line, and thus that IL-13R.alpha.2 is a cancer stem cell antigen.
This fact was verified in a subsequent duplicate experiment (see
FIG. 8). Moreover, as shown in FIG. 5, IL-13R.alpha.2 is was
expressed to higher levels on on CD133+Cells as compared to CD133-
cells.
6.1.3 Conclusion
[0156] These data demonstrate that EphA2 is a cancer stem antigen,
and thus can be used in methods for the treatment of cancer, such
as brain cancer.
EQUIVALENTS
[0157] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims.
[0158] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
Sequence CWU 1
1
519PRTArtificial Sequencepeptide derived from human EphA2 protein
1Thr Leu Ala Asp Phe Asp Pro Arg Val1 5 29PRTArtificial
Sequencepeptide derived from human IL-13R-alpha2 protein 2Trp Leu
Pro Phe Gly Phe Ile Leu Ile1 5 39PRTArtificial Sequencepeptide
derived from human IL-13R-alpha2 protein 3Trp Leu Pro Phe Gly Phe
Ile Leu Val1 5 49PRTArtificial Sequencepeptide derived from human
IL-13R-alpha2 protein 4Ala Leu Pro Phe Gly Phe Ile Leu Val1 5
59PRTArtificial Sequencepeptide derived from human IL-13R-alpha2
protein 5Glu Leu Pro Phe Gly Phe Ile Leu Val1 5
* * * * *